49TH BIOPHYSICAL SOCIETY ANNUAL MEETING ADDENDUM & LATE ABSTRACTS

FEBRUARY 12–16, 2005, LONG BEACH, CALIFORNIA

SUNDAY POSTER SESSIONSPosters Not being Presented

812-POS BOARD #B651Terbium Cation Luminescence as a Probe of Solvent Shell Properties with-in both Glass and Sol-gel Matrices. Sarah J. Paquette, J.M. Friedman.

830-POS BOARD #B669The Mechanical Properties of Polycystin-1 Ectodomain. AndresOberhauser, F. Qian.

840-POS BOARD #B679Chondroitin Sulfate Proteoglycan Conformation and Mechanical Properties: A Molecular Modeling Investigation. Mark Bathe, A.J. Grodzinsky, G.C. Rutledge, B. Tidor.

855-POS BOARD #B694An Intermediate-level Uncergraduate Course on Biological Physics. Philip Nelson.

— MONDAY, FEBRUARY 14 —

— SUNDAY, FEBRUARY 13 —

1

MONDAY POSTER SESSIONSAbstract did not appear in Abstracts Issue

Molecular Dynamics Study of Nucleotide Gated Ion Channel Activity ofAquaporin-1. Jin Yu, Klaus Schulten, Emad Tajkhorshid. University ofIllinois at Urbana-Champaign, Urbana, IL, USA.Aquaporin-1 (AQP1) is a member of aquaporin family of water channels thatare present in all life forms. Although a single aquaporin protein forms a com-plete transmembrane water pore, aquaporins exist as tetramers, with a struc-tural organization reminiscent of ion channels, such as K and CNG channels.It has been proposed, and experimentally shown, that the tetrameric (central)pore of AQP1 can conduct cations upon cGMP activation. Starting with thehigh-resolution structure of AQP1 we have investigated the molecular mecha-nism of cGMP-gated ion conduction through AQP1's central pore withmolecular dynamics (MD) simulations. We induced an ion permeation eventby constraining a sodium ion at different regions along the pore, and investi-gated the induced protein conformational changes, which are likely to be relat-ed to those involved in the actual gating of the channel. The most prominenteffects were hydration of the channel, and the large movement of a cytoplas-mic loop, consistent with its high mobility as inferred from different crystalstructures of AQP1. In order to simulate the binding of cGMP and to studyhow it might trigger the gating of the central pore, we placed four cGMP mol-ecules on the cytoplasmic side of the AQP1 tetramer at locations far from thecentral pore, and monitored their motions during the simulations.Interestingly, two of the nucleotides diffused towards the central pore along acluster of arginine residues, and established direct contacts with the mobilecytoplasmic loop described above. Comparison with control simulations showsthat cGMP increases the magnitude of the observed conformational changes.We have also mutated key residues lining the constriction region of the chan-nel. In comparison with wild-type behavior, these mutations can increase theopen probability of the channel, a property that can be experimentally tested.

MONDAY POSTER SESSIONSPosters Not being Presented

1190-POS BOARD #B177Dynamics and Relaxations in Wrinkling of Lipid Membranes. Ahmed Abd El Rahman, S. Chaieb.

1213-POS BOARD #B200Single-Molecule Fluorescence Characterization of Bilayers Suspended OverMicrodrilled Holes in Glass. Robert A. Horton, J.C. Conboy, J.M. Harris.

1651-POS BOARD #B637Live Cell Imaging of Actin and Tubulin Dynamics by Multiple FRET Detection.Yusuke Niino, T. Iguchi, K. Oka.

— FRIDAY, FEBRUARY 11 —FRIDAY SATELLITE MEETING

Complete Speaker Information not included in ProgramDrug Discovery for Ion Channels V

SpeakersIan M. Herzberg, ALA Scientific InstrumentsDaniel Bertrand, University of Geneva, SwitzerlandArthur M."Buzz" Brown, ChanTest, Inc.Steffen Hering, University of ViennaMads P.G. Korsgaard, NeuroSearch A/SCharles W. Luetje, University of MiamiJoe McGivern, AmgenUmesh Patel, Cytomyx Ltd.Gail A. Robertson, University of Wisconsin, MadisonAllessandro Taddei, Axxam srl.D.J.Trezise, GSK Medicines Research CentreGerhard Trube, Hoffmann - La Roche Ltd.Alan Verkman, University of California, San FranciscoCaterina S. Virginio, GlaxoSmithKlineKeWei Wang, Wyeth ResearchAlan Finkel, Molecular Devices

MONDAY SPECIAL EVENTSSpeaker Added

Education Committee Panel: Innovations in the Teaching of Biophysics

Workshops in the Chemical Sciences for College and University Teachers. Jerry Smith, Georgia State University.

MONDAY PLATFORM SESSIONSContributing Authors Omitted in Program

Platform WOther Ligand-gated Channels

923-PLAT 11:45 AMConformational Flexibility of a Proline Peptide Bond in the M2-M3 Loop of the 5-HT3 Receptor Controls Channel Opening. Sarah C.R. Lummis1, D.L. Beene1,H.A. Lester2, D.A. Dougherty1, 1Chem. and Chem. Engin, Caltech, Pasadena, CA,USA; 2Biology, Caltech; 3Biochemistry, Univ of Cambridge, Cambridge, UnitedKingdom.

— TUESDAY, FEBRUARY 15 —TUESDAY WORKSHOP

Changed Talk TitleWorkshop 4

Advances in Single-Molecule and Single-Cell Detectionand Manipulation

1884-WKSHP 8:42 PMDirect Detection of Individual Basepair Steps during TranscriptionalElongation by RNA Polymerase. Steven Block, Stanford University.

TUESDAY PLATFORM SESSIONSWithdrawn Speaker

1804-PLAT 11:00 AMSpontaneous Formation of DHPC Micelle Around the Outer Membrane ProteinOmpX. Rainer A. Böckmann, A. Caflisch, Univ Zürich, Switzerland.

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— WEDNESDAY, FEBRUARY 16 —

— EXHIBITS —Title of Exhibitor Presentation Changed

1:30 PM–3:30 PMSupercharging Automated Patch Clamp for Ion Channel Screening.Alan Finkel, Molecular Devices Corporation

Exhibits not Listed in ProgramBeckman Coulter, Inc. 22011800 S.W. 147th AvenueMiami, FL 33196305-380-3800www.beckmancoulter.comThe Multisizer 3, based on the Coulter Principle, provides the highest resolution avail-able for counting and size distribution analysis of cells larger than 0.4 um. It can dis-criminate between two cells close in size better than any other instrument. TheMultisizer 3 is the only instrument capable of directly measuring cell volume changesin real time. This is possible thanks to the use of a Digital Pulse Processor (DPP).

DVC Company 53510200 Highway 290 WestAustin, TX 78736512-301-9564www.dvcco.comThe new DVC-4000M (2048x2048) & DVC-2000 (1600x1200) camera series willbe demonstrated at DVC's booth. Both 12-bit cameras are based on high-QE KodakCCDs with 7.4µ square pixels. Both cameras have low read noise, ideal for quantita-tive applications. Adjustable binning, ROI and asynchronous pulsed exposure opera-tions are supported via OEM-friendly software. TE-cooling is optional and retro-fittable.

Oxford University Press 4362001 Evans RoadCary, NC 27513919-677-0977www.oupjournals.orgOxford University Press is a department of the University of Oxford. It furthers theUniversity's objective of excellence in research, scholarship and education by publish-ing worldwide. OUP publishes top journals in the field of biophysics, such asMolecular Biology and Evolution, Biostatistics, Journal of the ICRU, and RadiationProtection Dosimetry.

Tecan U.S. 737PO Box 13953Durham, NC 27709800-352-5128www.tecan.comTECAN, with a 24-year history in the liquid handling and microplate processing busi-ness, is synonymous with outstanding performance, efficiency and superior technicalsupport and service. TECAN offers a wide selection of liquid handling platforms,microplate readers/washers and microarray instrumentation to address many of theprofound challenges facing biomedical laboratories today.

Changed Booth NumberCalorimetry Sciences Corp.OLD BOOTH NUMBER: 410NEW BOOTH NUMBERS: 239 & 241

* The Biophysical Society gratefully acknowledges the educational grantprovided by Merck Research Laboratories.

WEDNESDAY PLATFORM SESSIONSWithdrawn Speakers

Platform BFTranscription & Chromatin

2666-PLAT 11:45 AMAcoustic Wave Network Analysis and Signaling of Gene Transcription in the On-line Format. Christine N. Jayarajah, M.Thompson, Univ Toronto.

Platform BKPeptide & Toxin Ion Channels

2709-PLAT 12:30 PMSpin Labeling EPR Study of Indolicidin and Toac-Labeled Indolicidin-MembraneInteraction. Shirley Schreier, R.F.F. Vieira, E.F. Poletti, C.R. Nakaie, A. Lucia C.F.Souto, Inst Chem, Univ São Paulo, Brazil; Fed Univ São Paulo.

WEDNESDAY POSTER SESSIONSRescheduled Poster

2757-POS BOARD #B40Application of the Planck-Benzinger Thermal Work Function in InteractingBiological Systems. Paul W. Chun. RESCHEDULED TOTUESDAY POSTER SESSIONS2757-POS BOARD #R2

Withdrawn Posters2962-POS BOARD #B242Molecular Basis of Scorpion Alpha-Toxins Preference for Rat Brain or InsectSodium Channels. I. Karbat, N. Ilan, F. Frolow, O. Froy, L. Cohen, R. Khan, N. Gilles, M. Turkov, M. Benveniste, Dalia Gordon, M. Gurevitz.

Authors not listed in Program2982-POS BOARD #B262Modulations of HERG K+ Channel Function by PKA and PKC Activators andInhibitors: Direct Actions and Indirect Mediation by Reactive Oxygen Species. J. Wang, Huixian Lin, Y. Zhang, Z. Wang.

2997-POS BOARD #B277Physiological Function of Herg K+ Channels in Cardiac Repolarization as Reported by Action Potential-Clamp Techniques. Xiaobin Luo, Y. Zhang, J. Wang, H. Lin, Z. Wang.

3257-POS BOARD #B537Development of Combined AFM-Single Molecule Fluorescence Strategies for theStructural and Mechanistic Characterization of Multiprotein-DNA Complexes.Robert D. Brodnick, I. Tessmer, D.A. Erie, E. Borgerding.

Author Scheduled for both presentation timesPRESENTING AT 1:45 PM3227-POS BOARD #B507Dark Fraction and Blinking of Water-Soluble Quantum Dots in Solution.Jie Yao, D.R. Larson, W.R. Zipfel, W.W. Webb.

PRESENTING AT 2:45 PM2796-POS BOARD #B77Multiphoton Imaging of Drosophila Polytene Nuclei in Live Cells for Transcription Study. Jie Yao, J.T. Lis, W.W. Webb.

TUESDAY POSTER SESSIONSPosters Not being Presented

1945-POS BOARD #B57Oligomerization and Aggregation of Ribonuclease A Induced by Heat. Yong-Bin Yan, J. Zhang, H-W. He, H-M. Zhou.

2090-POS BOARD #B202Composition of Supported Model Membranes During Phospholipase A2Hydrolysis. Hanna P. Vacklin, F. Tiberg, G. Fragneto, R.K. Thomas.

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POS-L1 BOARD#LB1Quantitative Measurement of Amino-Acid Side Chain Accessibility in MembraneProteins: Results of a New Pulse-Chase Method Applied to Bovine Rhodopsin. JosephS. Schoeniger, MD, PhD, Kenneth Sale, PhD, Richard B. Jacobsen, Marites Ayson.Sandia National Laboratories, Livermore, CA, USA.Measurement of the solvent accessibility of amino acid side chains in a protein can giveinsight into protein structure, dynamics and ligand association. Solvent accessibility meas-urements can be made by many methods (NMR, EPR, fluorescence, Mass Spectrometry(MS)). We have recently shown that MS methods, which require only minute amounts ofprotein, can be used to rapidly profile the relative reactivity towards various alkylatingreagents of side chains in wild-type proteins. The measured reactivity, however, combinessolvent accessibility and properties such as nucleophilicity (i.e., pKa). Further, MS results,though useful for estimating relative order of reactivity, are non-quantitative. We presenthere quantitative measurements of first-order rate constants of lysine and cysteine residuesin native Bovine Rhodopsin in Rod-Outer-Segment (ROS) vesicles reacting with alkylationreagents. The rate constants are obtained by non-linear-least-squares fits of data obtainedfrom multiple pulse-chase experiments. Product abundance levels are measured by LiquidChromatography/Electrospray MS (LC-ESI-MS) of Rhodopsin CNBr peptides. Pulse andchase reagents are such that alkylated peptide products have essentially identical protonaffinities: the relative intensities are quantitative. When we compare the rate constants tosolvent accessibilities calculated from the Rhodopsin crystal structure, we find that moreaccessible residues react more rapidly. However, we also find that a small fraction of someresidues in the ROS are non-reactive. The quantitative measurement of reaction rate con-stants using MS forms the foundation for new studies of the biophysical chemistry of mem-brane and other “difficult” or low-abundance proteins in their native membrane/environ-ment: for example, measurement series at different temperature or pH or could resolve,respectively, activation energies or contributions to reactivity of pKa versus accessibility.Finally, the method is extensible to other residue types (Y,E,D) using existing chemistries.Author Disclosure Block: J.S. Schoeniger, None; K. Sale, None; R.B. Jacobsen, None; M.Ayson, None.

POS-L2 BOARD #LB2Artificial Allosteric Control of Proteins. Brian Choi, Giovanni Zocchi, Jeanne Perry,Sum Chan, Yim Wu. University of California, Los Angeles, Los Angeles, CA, USA.We built an artificial mechanism of allosteric control of a protein, based on mechanicalstress. The Maltose-Binding Protein (MBP) of E. coli undergoes a conformational changeupon binding maltose. Introducing a mechanical stress favoring one or the other confor-mation will therefore alter the binding affinity for the substrate. We have constructed achimera where the two lobes of the maltose binding protein are covalently coupled to theends of a DNA oligomer. The mechanical tension on the protein is provided by the bend-ing elasticity of the DNA, and is controlled by exploiting the difference between singlestranded and double stranded DNA. We report that the binding affinity of MBP for malt-ose is significantly altered by the tension, which was varied by allowing DNA oligomers ofvarious lengths to hybridize to the DNA of the chimera. By the same method, we controlthe enzymatic activity of a second protein: Guanylate Kinase from MycobacteriumTuberculosis. This study exemplifies a general strategy to introduce artificial control ele-ments in the function of proteins. Author Disclosure Block: B. Choi, None; G. Zocchi, None;J. Perry, None; S. Chan, None; Y. Wu, None.

POS-L3 BOARD #LB3Acetylcholine induced conformational changes in acetylcholine-binding proteinreviewed by MD simulation, Trp fluorescence and TROSY type NMR. Fan Gao1,Georges Mer1, Nina Bren1, Scott Hansen2, Palmer Taylor2, Steven M. Sine1. 1MayoGraduate School, Rochester, MN, USA, 2Univ. of California, San Diego, San Diego,CA, USA.We delineate acetylcholine (ACh) dependent conformational changes in acetylcholine-binding protein (AChBP, 125kDa), a prototype of the nicotinic receptor ligand bindingdomain, by using molecular dynamics (MD) simulation, intrinsic tryptophan (Trp) fluo-rescence and 2D NMR spectroscopy. MD simulation of AChBP shows that binding ofACh establishes close register of Trps from adjacent subunits, W143 and W53, and drawsthe peripheral C-loop inward to occlude the entrance to the binding cavity. Close registerof W143 and W53 is demonstrated by ACh-mediated quenching of intrinsic Trp fluores-cence, elimination of quenching by mutation of one or both Trps to Phe, and decreasedlifetime of Trp fluorescence by bound ACh. The inward motion of C-loop is demonstrat-ed by using TROSY type 2D NMR, which shows an ACh-mediated change in chemicalshift of isotopically-labeled vicinal cysteines at the tip of the C-loop. The collective findingsshow that ACh initially establishes close register of conserved Trps from adjacent subunitsand then draws the C-loop inward to occlude the entrance to the binding cavity. AuthorDisclosure Block: F. Gao, None; G. Mer, None; N. Bren, None; S. Hansen, None; P.Taylor, None; S.M. Sine, None.

POS-L4 BOARD #LB4Hydration of Hydrophobic Cavities: A Molecular Dynamics Study of HumanInterleukin-1Beta. Sandeep Somani, Choon-Peng Chng, Chandra S. Verma,Bioinformatics Institute, Singapore, Singapore.Hydrophobic cavities in proteins are generally assumed to be empty since x-ray crystallog-raphy does not reveal any buried waters (unless at least one hydrogen bond can be formed).However, in the protein human Interleukin-1beta [HIL], NMR (NOE) observations sug-gested, and later X-ray crystallography confirmed the presence of waters in a totallyhydrophobic cavity. However, the precise number of water molecules, their pathways ofexchange with bulk water and the associated residence times have yet to be characterized.To this end we have carried out a series of molecular dynamics simulations. The hydropho-

bic cavity is large enough to accommodate up to four water molecules. This number wasvaried between 1 and 4, and the dynamics simulated. The simulations showed, that inagreement with NMR, the waters do reside within the cavity for periods exceeding 1 ns(this being the minimum residence time required to lead to a detectable NOE). Further,the study shows that there is considerable positional disorder within the cavity whichexplains the lack of detection by crystallography. Our study also identified pathways inaddition to those evident from analyses of static crystal structures. Finally, analyses of thehydrogen-bond network of waters inside the cavity shows a propensity to form dimers orwater rings made up of 4 waters. Author Disclosure Block: S. Somani, None; C. Chng, None;C.S. Verma, None.

POS-L5 BOARD #LB5Enzyme Dynamics of a Hyperthermophilic Adenylate Kinase Studied byCrystallography, NMR and Computation. Vu T. Thai, Dorothee Kern. Brandeis University, Waltham, MA, USA.Conformational flexibility in proteins is known to facilitate events such as substrate recog-nition, catalysis, and allosteric transitions. However, structures of macromolecules solved byx-ray crystallography usually depict only one conformational substate. Here, we present thecrystal structures of a hyperthermophilic adenylate kinase from Aquifex aeolicus (Aadk) inthe apo form and in complex with the bi-substrate analog Ap5A. The apo structure con-tains three distinctly different molecules in the asymmetric unit. Each molecule differs inthe orientations of the ATP- and AMP-lids. Superposition of the three apo conformationsonto the Ap5A bound structure shows that the lid motions are on path toward the fullyclosed conformation. 15N NMR relaxation dispersion experiments were performed on apoAadk in order to determine whether the protein undergoes conformational exchange insolution. The dynamic hot spots located by NMR correspond well to regions of the pro-tein that must move in order to interchange between the different forms observed in thecrystal structure. Computational analysis of Aadk flexibility highlights these same regions,as well as predicts the motions exhibited by the substates. The rate of exchange for theseresidues has been quantified using the NMR data and can be fit with a single global process.The data presented here shows how a combined approach between crystallography, NMRand computation can further advance our understanding of protein motions. AuthorDisclosure Block: V.T. Thai, None; D. Kern, None.

POS-L6 BOARD #LB6Conformational Dynamics of Phototropin LOV2 Domain Studied by NMRRelaxation Dispersion Measurement. Xiaolan Yao, Kevin Gardner, Michael Rosen.Univ. of Texas Southwestern Medical Center, Dallas, TX, USA.Phototropins are a group of light-activated kinases responsible for a variety of light-regulat-ed activities in plants. LOV2 (light-oxygen-voltage) domain is the photo-sensor of pho-totropins. Previous solution NMR studies on Avena Sativa phototropin LOV2 domain(AsLOV2) indicate that the C terminal helix in AsLOV2, designated Jα, loses its helicalconformation upon absorbance of blue light (446 nm) and becomes a random coil. Thisprocess is reversible on tens of second time scale. Solution NMR studies also show that Jαis rather dynamic even in the dark state. Therefore it is very likely LOV2 exists as equilib-rium of two states: Jα bound to the LOV2 core and J? unbound to LOV2 core. This typeof conformational exchange in protein normally occurs on the time scale of seconds andmilliseconds. In this work, we used NMR Carr-Purcell-Meiboom-Gill (CPMG) relaxationdispersion measurements to probe the conformational dynamics of dark-state AsLOV2.Backbone 15N and methyl 13C CPMG experiments are performed at temperatures rang-ing from 5ºC to 25ºC. Sites showing dynamics are mainly in the Jα and the Jα-LOV2 coreinterface. The exchange rates, excited state populations and the chemical shift differences ofthe two states are obtained from fitting of the experimental data. The free energy changesassociated with this process are obtained from measurements at different temperatures.Author Disclosure Block: X. Yao, None; K. Gardner, None; M. Rosen, None.

POS-L7 BOARD #LB7Conformational Transitions of Flexible Protein and its Effects on Kinetics of Protein-Protein Binding Interactions. Jennifer Myphuong Bui, BS, J. Andrew McCammon,PH D. UC-San Diego, La jolla, CA, USA.Conformational state of a protein depends on the relative thermodynamic stabilities of thevarious accessible conformations and on the kinetics of their interconversion.Characterizing transitions between different conformational states is important in under-standing mechanisms underlying protein-protein interaction process. Fasciculins (FAS),61-amino-acid peptides, are pico-molar protein inhibitors of synaptic acetylcholinesterases.Four fasciculins have been characterized to date: FAS1 and FAS2 from the venom ofDendroaspis angusticeps, FAS3 from the venom of D.viridis, and toxin C from the venomof D.polylepis. Although the primary structural sequences of FAS1 and FAS2 differ by onlyone residue at position 47, two distinct stable conformers of FAS1 and FAS2 have beencrystallized. In this work, two 0.2- microsecond molecular dynamics (MD) trajectories ofFAS1 and FAS2 are carried out to sample the conformational space accessible to the sys-tems. Protein-protein interactions and complexation between FAS and acetylcholinesteras-es have also been modeled The MD results are analyzed using principal component analy-sis on distance matrices, cluster analysis, and compared with those obtained from a 15-nanosecond MD simulation of FAS bound to acetylcholinesterase in order to identifyimportant structural transitions upon complexation. Conformational transitions betweenFAS1 and FAS2 are also characterized to gain insights into their effects on the kinetics ofbinding to acetylcholinesterases. Author Disclosure Block: J.M. Bui, None; J. McCammon,None.

POS-L8 BOARD #LB8Low frequency dielectric spectroscopy using a nanogap sensor to detect conformationalchanges of ferricytochrome c in aqueous solution. Joshua T. Nevill, PhD in progress,Dino Di Carlo, Peng Liu, Ki-Hun Jeong, Luke P. Lee. UC Berkeley, Berkeley, CA, USA.We have developed a unique electrode-based biosensor that uses dielectric spectroscopy tointerrogate samples inside a nanoscale gap, and we have shown that this biosensor is sensi-tive enough to record the conformational changes of cytochrome-c.Dielectric spectroscopy (DS) offers an excellent detection platform for molecular scale

— LATE ABSTRACTS —1:00—3:00 PM, HALL A/B

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biosensors because it is label-free and can be used with aqueous solutions. DS is a wellknown technique, but low frequency DS (<1 MHz) has been avoided because of dominat-ing signals associated with both electrical double layers and solution conductivity. The sig-nals due to subtle changes in ionic strength that overwhelm traditional measurements areabsent in our device, which we propose is because the electrode gap size (20-100 nm)approaches the Debye length.Although low frequency DS is not conventionally used, the potential for using this tech-nique to study macromolecule conformation is substantial, since the dielectric response ofslowly relaxing macromolecules can be examined. Additionally, the sizes of these cavitiesapproach the same scale as the molecules themselves, which could provide insight that waspreviously unattainable with traditional setups.Cytochrome-c was chosen as the experimental model because it is well characterized. Werecorded a pH-dependent transition in dielectric properties below 1 MHz that is consistentwith previously observed conformational changes. Kramers-Kronig relations were employedas a means of checking the validity of dielectric spectroscopy results, which show thatnanogap biosensors are better at providing accurate and conclusive results than standardsetups. The results demonstrate that this nanogap sensor presents a powerful potential plat-form for identifying conformational states of proteins. These biosensors could also providean excellent platform for high throughput proteomic chips, since they can be fabricated informats that are amenable to high density arrays. Author Disclosure Block: J.T. Nevill, None;D. Di Carlo, None; P. Liu, None; K. Jeong, None; L.P. Lee, GlaxoSmithKline Research grants;Intel Research grants.

POS-L9 BOARD #LB9Hierarchical Coarse-Graining for Large Protein Dynamics. Chakra S. Chennubhotla,Ph. D., Ivet Bahar. University of Pittsburgh, Pittsburgh, PA, USA.Elastic network models have been successful in elucidating the fluctuation dynamics of pro-teins around their native conformation. The network models use alpha-carbons of the fold-ed proteins as nodes of a mass-spring system, where each node is connected by springs (ofuniform spring constant) to all other neighboring nodes located within a cutoff distance(encompassing at least the first coordinate shell around each residue). It has been observedthat dominant motions of protein structures can be obtained by further coarse-graining ofthe protein structure along the backbone. However, this procedure raises several implemen-tation issues including the proper scaling of the parameters, spring constant and cutoff dis-tance, for coarse-graining.To this end, we propose a novel graph-theoretic approach to generating a hierarchy ofcoarse-grained structures. In particular, we consider proteins as weighted undirected graphsand undertake random walks on such graphs. The Markov chain propagation causes infor-mation at neighboring nodes to become less distinguishable. This in turn allows for a coars-ening procedure to be applied on the graph. In fact, we show how to generate a hierarchyof coarse-grained structures from either a fully atomistic description or a backbone repre-sentation of the protein. The adjustment of the spring constant and the cutoff distance alongthe hierarchy is automatic. Additionally, for large targeted structures the reduction in com-putational time for elucidating the dynamics is significant. Author Disclosure Block: C.S.Chennubhotla, None; I. Bahar, None.

POS-L10 BOARD #LB10NMR characterization of conformation of polyubiquitin chains: insights into structuralbasis of specificity in ubiquitin-mediated signaling. Ranjani Varadan1, Michael Assfalg1,Olivier Walker1, Aydin Haririnia1, Shahri Raasi2, Cecile Pickart2, David Fushman1.1University of Maryland, College Park, MD, USA, 2Johns Hopkins University, Baltimore,MD, USA.Polyubiquitin chains function as signaling molecules that mediate a variety of cellularprocesses. Signaling by polyubiquitin (polyUb) involves the covalent modification of thesubstrate protein by one or more ubiquitin molecules linked via a specific lysine residue.Remarkably, polyUb chains linked via different lysines commit the substrate protein intodifferent cellular pathways. Although it has been anticipated that diversity in ubiquitin-mediated signaling arises from linkage-specific conformational differences between alterna-tively linked polyubiquitin chains, direct structural evidence in support of this view has beenlacking. Therefore, knowledge of the conformation of differently linked ubiquitin chainswill elucidate any such linkage specific differences and help in understanding the molecularbasis of specificity in Ub-mediated signaling.Using several NMR methods, we characterized the conformation of Lys48- and Lys63-linked Ub2 chains. We show that Lys48-linked Ub2 adopts a predominantly ‘closed’ con-formation, with a well-defined interface formed between the hydrophobic surfaces of thetwo Ub domains. In contrast, Lys63-linked Ub2 molecules are characterized by an extend-ed conformation with no definitive interface between the two Ub domains. The findingsare the first experimental demonstration that differently linked polyUb chains adopt distinctconformations in solution.Our binding studies reveal that, consistent with an extended conformation of the Lys63-linked Ub2, the hydrophobic surfaces on both Ub in this chain can bind ligands independ-ently. We find that, although these hydrophobic residues are sequestered at the interface inLys48-linked Ub2, they are rendered accessible for direct recognition because the Ub2 con-formation is in dynamic equilibrium between ‘open’ and ‘closed’ states. Further studies willelucidate the mode of recognition of Lys48-linked Ub2 chains, and help understand howdifferences in conformation of polyUb chains modulate their specific interaction with Ub-binding proteins. Author Disclosure Block: R. Varadan, None; M. Assfalg, None; O. Walker,None; A. Haririnia, None; S. Raasi, None; C. Pickart, None; D. Fushman, None.

POS-L11 BOARD #LB11Titration of His55 at the Dimer Interface of Dynein Light Chain LC8 is Coupled toDimer Dissociation. Elisar Barbar, PhD, Loren Cochrun, Afua Nyarko. Oregon StateUniversity, Corvallis, OR, USA.Dynein light chain LC8 is a highly conserved light chain subunit of cytoplasmic dynein thatinteracts with a wide variety of proteins and is thus thought to play a fundamental role inboth the assembly of the motor complex and the recruitment of cargo. LC8 is a stable dimerat physiological pH and dissociates to a folded monomer at lower pH with a titration mid-

point of 4.8 as determined from sedimentation equilibrium at different pH values. We havesuggested that a protonation of a histidine residue at the dimer interface, His 55, causes arepulsive interaction with His 55’ from the other subunit resulting in dissociation of thedimer. In this work we use 13C-1H NMR to determine the pKa’s of the three histidineresidues in LC8, His 72, His 68, and His 55. His 72 and 68 are close to the dimer inter-face but not stacked against each other as His 55 is. Site-directed mutagenesis of histidine55 to lysine resulted in a monomeric form and allowed determination of pKa’s of His 68and 72 in the monomeric form of LC8. Mutation of histidine 55 to alanine resulted in adimeric form and allowed comparison of pH dependence in the dimeric species. The resultsconfirm our earlier suggestion that titration of His 55 is coupled to LC8 monomer dimerequilibrium. Author Disclosure Block: E. Barbar, None; L. Cochrun, None; A. Nyarko,None.

POS-L12 BOARD #LB12Biophysical studies of the amino terminal domain of the sodium-channel MEC-4:inducible folding of a molten globule in vitro. Mark A. Arbing, Madhav Sukumaran,John F. Hunt. Columbia University, New York, NY, USA.The DEG/ENaC superfamily of ion channels mediates sodium transport across cell mem-branes, thus playing a critical role in many physiological processes. The MEC system, a sub-group of the degenerin (DEG) family, transduces the sensory response to touch in neuronsof C. elegans. The intracellular domains of the channel-forming subunits (MEC-4 andMEC-10) are involved in subunit assembly and channel gating. Our biophysical studies ofthe intracellular domain of MEC-4 have revealed that it forms a stable molten globule insolution possessing some secondary structure but lacking well ordered tertiary structure.The lack of well ordered structure suggested that it may require interaction with proteina-ceous or lipid partners to adopt a functional tertiary structure. A significant increase in theα-helical content of the domain was observed in circular dichroism experiments in the pres-ence of the detergents LDAO (lauryldimethylamine-N-oxide) and n-octyl-β-D-glucoside,and to a lesser extent with phospholipid analogues. Intrinsic tryptophan fluorescence spec-troscopy studies demonstrated an increase in total fluorescence consistent with thesereagents inducing a less solvent-exposed and more folded structure. However, the amountof ?-helical structure induced by detergent is less than that predicted from sequence analy-sis suggesting that the domain is not completely folded. Moreover, the fact that detergentsinduced more structure than phospholipids suggests the detergent effect is non-physiolog-ical. We therefore conclude that protein-protein interactions are likely to regulate the fold-ing and thereby the function of the N-terminal domain of the MEC-4 and MEC-10ENaC channels. Author Disclosure Block: M.A. Arbing, None; M. Sukumaran, None; J.F.Hunt, None.

POS-L13 BOARD #LB13Protein formulation of an IgG1 antibody with atypical glycosylation: a study on thestability of its glycoforms. Grace C. Chu, Ph.D1, Sururat Bamigbaiye Coulibaly, M.S.1,Artem Raibekas2, Songpon Deechongkit, Ph.D1. 1Amgen, Inc., Thousand Oaks, CA, USA, 2University of Toronto, Toronto, ON,Canada.One major challenge in developing protein formulation for antibodies is heterogeneity,which can arise from glycosylation, disulfide linkages, and lysine variations. Whether or notthe resulting isoforms have similar stability is a concern in protein formulation. Here we

discuss liquid formulation of animmunoglobulin, IgG1, with glycosyla-tion in both Fc and Fab. At least eightglycoforms were detected in the mam-malian-expressed filtered purified bulkmaterial (FPB) by cation exchange(CEX) chromatography (Fig. 1). Five ofthe glycoforms, Peaks -2 to 2, were iso-lated and placed on stability along withthe FPB at different temperatures andpH over time. Utilizing CEX, reversedphase, and size exclusion HPLC, as wellas fluorescence and circular dichroism

spectroscopy, we characterized the stability of each isoform and FPB for up to three months.The stressed glyocoforms and FPB showed differences in aggregate formation, cleavages,and structure. Author Disclosure Block: G.C. Chu, Amgen, Inc. Employment (full or part-time); S.B. Coulibaly, Amgen, Inc. Employment (full or part-time); A. Raibekas, None; S.Deechongkit, Amgen, Inc. Employment (full or part-time).

POS-L14 BOARD #LB14Nucleation events in amyloid beta-protein monomer folding studied in silico.Jose M. Borreguero1, Brigita Urbanc1, Noel Lazo2, Sergey V. Buldyrev3, David B.Teplow2, Eugene Stanley1. 1Center for Polymer Studies and Physics Department, BostonUniversity, Boston, MA, USA, 2Center for Neurologic Diseases, Brigham and Women’sHospital, and Department of Neurology, Harvard Medical School, Boston, MA, USA,3Department of Physics, Yeshiva University, New York, NY, USA.Oligomeric assemblies of the amyloid beta-protein (Abeta) have been mplicated in thepathogenesis of Alzheimer’s disease as a primary source of neurotoxicity. Recent in vitrostudies have suggested that a ten-residue segment, Abeta(21-30), forms a turn-like structurethat nucleates the folding of the full-length Abeta protein. To gain mechanistic insight intothe folding of Abeta(21-30) and to determine the structure of its folded state, we simulateAbeta(21-30) folding using a discrete molecular dynamics algorithm and a united-atommodel incorporating implicit solvent and variable electrostatic interaction strength.Independent of the electrostatic interaction strength, we find that Abeta(21-30) folds intoa loop-like conformation driven by an effective hydrophobic attraction between Val24 andthe butyl portion of the Lys28 side-chain. Varying the electrostatic interaction strength, we

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show that at medium strengths 1.5Kcal/mol, the class of unfolded monomer conforma-tions almost disappears, in agreement with experimental observations. Under conditionsoptimal for monomer folding, Glu22 and Asp23 form transient salt-bridges with Lys28that stabilize the loop conformations. Glu22-Lys28 is the most favored salt-bridge interac-tion. High electrostatic interaction strengths, which usually occur in the interior of proteins,destabilize the packing of Val24 and Lys28. Analysis of the unpacked structures reveals theformation of strong salt-bridges—-Glu22-Lys28 (23% frequency), Asp23-Lys28 (48%), orboth (29%). Our observations suggest that when the electrostatic interaction is strong, thesalt-bridge Asp23-Lys28 is more likely to form, in agreement with studies of molecularmodeling of full-length Abeta fibrils. The binary nature of salt-bridge formation by Lys28provides a mechanistic explanation for the linkage of amino acid substitutions at Glu22 andAsp23 with Alzheimer’s disease or cerebral amyloid angiopathy. Substitutions may alter thefrequency of Glu22 or Asp23 involvement in salt-bridge formation and affect the stabilityof the folding nucleus formed in the Abeta(21-30) region. Author Disclosure Block: J.M.Borreguero, None; B. Urbanc, None; N. Lazo, None; S.V. Buldyrev, None; D.B. Teplow,None; E. Stanley, None.

POS-L15 BOARD #LB15Buried hydrophobic stack residues are critical in the folding of the parallel beta-helix, amodel for the amyloid chain fold. Ryan Simkovsky1, Kristen Cook2, Jonathan King1.1Massachusetts Institute of Technology, Cambridge, MA, USA, 2University of CaliforniaSan Francisco, San Francisco, CA, USA.The parallel β-helix has been proposed as a model for the polypeptide chain conformationwithin amyloid fibers. To investigate sequence requirements for directing a polypeptidechain into a β-helical fold, we have carried out systematic, high-throughput mutationalstudies using the p22 tailspike. Residues 143-540 form the 13 rungs of tailspike’s β-helicaldomain. These exhibit extensive, ordered stacks of buried hydrophobic residues.Preliminary studies indicated that these stacked hydrophobic core residues play a critical rolein directing the formation of the β-helix. Single alanine mutations have been generated at145 sites in the β-helix, representing the entirety of the non-alanine buried core stackresidues, potential β-helix capping residues, and a number of solvent exposed control posi-tions. The in vivo folding and assembly of the mutant chains is being characterized at mul-tiple temperatures by SDS gel electrophoresis, which distinguishes the fully native, SDS-resistant state from partially folded or misfolded conformers. Alanine replacements at 86 ofthe 103 buried residues in the β-helix domain show serious defects in polypepetide chainfolding at physiological temperature, 37°C. In contrast, only 10 positions of the 76 residuescurrently characterized at 18°C, a permissive temperature for temperature sensitive foldingmutants, cannot fold to a native protein. These results indicate that a surprisingly large frac-tion of the buried hydrophobic stack residues are critical for β-helix folding at physiologi-cal temperature, while a much smaller subset of these residues represent positions trulyessential for guiding the folding process. Tight sequence control of each successive rung ofthe helix may be essential for correct folding, perhaps indicating a processive order in fold-ing. Supported by NIH GM17,980. Author Disclosure Block: R. Simkovsky, None; K.Cook, None; J. King, None.

POS-L16 BOARD #LB16Dissecting helix stabilizing effects of positively charged residues sited at the C-terminus.Sean M. Decatur, Ph.D., Yushuan Lai. Mount Holyoke College, S. Hadley, MA, USA.Alpha helices are characterized by backbone hydrogen bonding between the i residue car-bonyl and the i + 4 residue amide proton. The N and C-terminal ends of helices are thusless stable due to fewer intrahelical interactions. Certain functional groups and residues sta-bilize helix structure by capping the termini. While strong helix stabilizing effects have beendescribed for several amino acids and functional groups at the N-terminus, few helixenhancing C-terminal caps have been observed model helices, and the C-capping effect isgenerally regarded as much weaker than N-terminal capping. Some of the C-caps identi-fied include residues with positively charged side chains, such as lysine, D-arginine, and 2,3-diaminopropionic acid (Dpr).1,2 These groups are believed to stabilize the C-terminus viainteractions with the helix macrodipole as well as specific hydrogen bonding with C-termi-nal backbone groups.In this study we seek to determine the relative importance of electrostatic and hydrogenbonding interactions in helix capping by Dpr and D-arginine. Alanine rich peptides basedon the repeat (AAAAK)n are used as model helices. We have synthesized and studied thesepeptides ending with alanine, Dpr, lysine or D-arginine at the C terminus using FTIR andCD. 13C labeling of the N or C terminus of peptides with a C-terminal Dpr indicates thatDpr confers stability to the whole helix, but not to the C terminus specifically. Lysineincreases helix stability about as well as Dpr while D-arginine shows a lesser stabilizingeffect. These results suggest that electrostatic interactions play a larger role for the stabilizingeffects of positively charged C-capping residues.Footnotes1 Schneider, J.P. and DeGrado, W.F. (1998) J. Am. Chem. Soc. 120, 2764-2767.2 Deechongkit, S., Kennedy, R.J., Tsang, K.Y., Renold, P., and Kemp, D.S. (2000)Tetrahedron Letters 41, 9679-9683.Author Disclosure Block: S.M. Decatur, None; Y. Lai, None.

POS-L17 BOARD #LB17Repetitive Pulling Catalyzes Co-Translocational Unfolding of Barnase During ImportThrough a Mitochondrial Pore. Pu Tian, PhD, Ioan Andricioaei, PhD. University ofMichigan, Ann Arbor, MI, USA.We present a computational study of barnase unfolding during import into mitochondriathrough a model translocon. In contrast to thermal/chemical unfolding, the major inter-mediates of co-translocational unfolding are mainly mediated by non-native interactionsaccompanying the protein configurations induced by pulling forces. These energy contri-butions, combined with backbone topological constraints imposed by the model pore,result in milestones along the unfolding pathways which are significantly different not onlyfrom those experienced during thermal/chemical denaturation, but also from those

observed in single-molecule pulling by both ends without pore constraints. Two on-path-way major translocation intermediates trapped in long-lived states by significantly highunfolding barriers are identified. A fraction of these pathways can, however, skip such localkinetic traps and result in extremely fast translocations, leading to a dramatic kinetic parti-tioning spanning approximately 4 orders of magnitude. The fraction of fast translocationevents is shown to increase upon switching the pull off and on, when compared to pullingat constant force. This suggests a ``catalytic’’ mechanism by which the mithochondrialimport machinery speculates this partitioning by using repetitive pulling in cycles. AuthorDisclosure Block: P. Tian, None; I. Andricioaei, None.

POS-L18 BOARD #LB18QUEUEING AND COOPERATIVITY IN LIGAND-RECEPTOR BINDING.Maria D’Orsogna, Ph.D., Thomas Chou. UCLA, Los Angeles, CA, USA.We compare the kinetics of two types of receptors: receptors that onlyallow ligands to attach to the binding sites in a specific order, and those that allow bindingin any order. For equivalent rate constants and cooperativities, we find that receptors withsequential ligand binding are more likely to have all of its binding sites fully occupied thanreceptors that bind ligands in any order. The mean occupation of sequentially loaded recep-tors is also higher. However, starting from a totally empty receptor, we find that the meanfirst passage time to full occupancy is smaller for low-mean- occupation, random adsorp-tion receptors. Our results are contrasted with Hill-like descriptions of receptor occupancyand scenarios in which distinction of receptor types may be important are discussed. AuthorDisclosure Block: M. D’Orsogna, None; T. Chou, None.

POS-L19 BOARD #LB19Differentiating the Chemical Requirements for Ligand-Induced Allosteric Inhibitionfrom those of Ligand Binding; Phenylalanine Inhibition of Pyruvate Kinase. AronFenton, Ph.D. The University of Kansas Medical Center, Kansas City, KS, USA.Within the brain of individuals with phenylketonuria (PKU), increased Phe concentrationsinhibit pyruvate kinase (M1-PYK). In vitro M1-PYK is allosterically inhibited by Phe bind-ing at a site distinct from the active site. Thus, drugs that bind competitively with Phe butdo not allosterically inhibit PYK could represent a treatment for PKU. The Phe binding siteof M1-PYK has not been identified. Therefore, we have used rabbit M1-PYK and Pheanalogs to discriminate the chemical regions of the allosteric ligand important for elicitingthe allosteric effect from the regions important for binding. Of the 20 standard amino acids,Gly, Ala, Ser, Cys, and Thr all bind competitively with Phe but do not inhibit PYK activi-ty. Ile, Trp, Leu, Val, Met, and Pro all inhibited the substrate affinity of M1-PYK but bindwith lower affinities than Phe. No inhibition or binding was detected for the other standardamino acids. Using chemical analogs of both Phe and Ala, we show that the ethylaminesub-structure contained in Phe is primarily responsible for ligand affinity. Beyond the betacarbon, sequential additions of the hydrophobic carbon chain length (up to norleucine)increase the allosteric effect. Therefore, the length of the hydrophobic side chain of theallosteric ligand appears to be the primary requirement for eliciting the allosteric inhibitionof M1-PYK. Author Disclosure Block: A. Fenton, None.

POS-L20 BOARD #LB20Nonplanar Heme Deformations in Horseradish Peroxidase Detected by RamanSpectroscopy at Soret Excitation. Qing Huang, Ph.D, Reinhard Schweitzer-Stenner.Chemistry Department, Drexel University, Philadelphia, PA, USA.We studied the out-of-plane modes of horseradish peroxidase (HRP) in different spin, oxi-dation and ligation states by measuring the respective resonance Raman spectra with (near)Soret excitation. The out-of-plane modes of the heme are Raman inactive for planar macro-cycles, but become resonance Raman active in the presence of out-of-plane deformations.The, thus, induced Raman scattering results mostly from Franck-Condon type coupling.We observed bands from a variety of out-of-plane modes such as g5, g6, g7 (A2u) modes,g21 and g22 (Eg), g15 (B2u) and g11 (B1u). The appearance of these bands is clearlyindicative of nonplanar deformations of the same symmetry. We determined the relativeintensities of all sufficiently intense Raman modes of ferric pentacoordinated, quantummixed and hexacoordinated low spin as well as of ferrous pentacoordinated high spin HRP.The apparent vibronic coupling parameters were obtained from a self-consistent analysis ofthe Raman intensities and the respective optical absorption spectrum. They revealed signif-icantly reduced displacements of the excited B-state for the ferrous, deoxy-like state com-pared with the resting ferric state. An analysis of the obtained coupling strengths of out-of-plane modes revealed that they are predominantly induced by static nonplanar deforma-tions along the normal coordinates of the lowest frequency modes of a given symmetrytype. We used the deformations of the heme in resting HRP to determine the vibronic cou-pling strength of the experimentally detectable out-of-plane modes and subsequentlyemployed this information to obtain the non-planar distortions for the investigated ferrichexacoordinated low spin state. This yielded a substantial reduction of the ruffling distor-tion, whereas the doming deformation remained mostly unaffected by the change of theiron’s spin and ligation state. Author Disclosure Block: Q. Huang, None; R. Schweitzer-Stenner, None.

POS-L21 BOARD #LB21Probing Single-Molecule Enzymatic activity and Gene Expression in Live Cells. X.Sunney Xie. Harvard University, Cambridge, MA, USA.Probing Single-Molecule Enzymatic activity and Gene Expression in Live CellsBy integrating new biochemical probes with advanced microscopy, our group is able tomonitor individual turnovers of a single enzyme molecule and to detect protein moleculesgenerated in living cells one at a time. Author Disclosure Block: X. Xie, None.

POS-L22 BOARD #LB22PHOTOTHERMAL STUDIES OF CO PHOTODISSOCIATION FROMHUMAN HEMOGLOBIN. Randy W. Larsen, Ph.D., Joseph Ganley, B.S.. University of South Florida, Tampa, FL, USA.In this study we have constructed volume and enthalpy profiles for CO photo-dissociationand subsequent recombination in human Hemoglobin (HbA) using time resolved pho-tothermal methods and transient absorption spectroscopy. Both photoacoustic calorimtery

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(PAC) and photothermal beam deflection (PBD) indicate that dissociation of CO from thetetrameric protein exhibits a volume increase of 18 + 1 ml/mol with a correspondingenthalpy change of 13 + 4 kcal/mol. Interestingly, at the low power densities used in thesestudies only a single kinetic phase can be identified and corresponds to CO recombinationto the heme iron. Thus, additional teriary/quaternary changes associated with the R-T tran-sition are not observed. These results are also quite distinct from those observed for COrecombination to Mb in which a 700 ns phase is observed and has been attributed to saltbridge interactions between peripheral Arg residues and the heme priopionates. Such inter-actions also appear to be absent in Hb. Author Disclosure Block: R.W. Larsen, None; J.Ganley, None.

POS-L23 BOARD #LB2315N and 31P solid-state NMR spectroscopy of M2 protein transmembrane domain inhydrated cylindrical lipid bilayers confined to nanopores. Eduard Y. Chekmenev1,2,Jun Hu1, Peter L. Gor’kov1, William W. Brey1, Timothy A. Cross1,2, Andres Ruuge3,Alex I. Smirnov3. 1NHMFL, Tallahassee, FL, USA, 2Florida State University, Tallahassee,FL, USA, 3North Carolina State University, Raleigh, NC, USA.Polarization inversion spin exchange at the magic angle (PISEMA) solid-state NMR exper-iment that correlates anisotropic dipolar and chemical shift interactions is now widely usedin structural and functional studies of oriented membrane proteins. We present high reso-lution 15N 2D PISEMA NMR spectra of a transmembrane peptide aligned using hydrat-ed cylindrical lipid bilayers formed inside nanoporous anodic aluminum oxide (AAO) sub-strates (Chekmenev, et al., 2005). The transmembrane domain of M2 protein frominfluenza A virus was reconstituted in hydrated 1,2-dimyristoyl-sn-glycero-3-phosphatidyl-choline bilayers that were macroscopically aligned by a conventional micro slide glass sup-port or by the AAO nanoporous substrate. 15N and 31P NMR spectra demonstrate uni-form alignment in the nanopores of both the phospholipids and the protein transmem-brane domain.Nanoporous AAO substrates offer advantages for membrane protein alignment in solid-state NMR studies compared to conventional methods. Specifically, excellent accessibilityto the bilayer surface for exposure to solute molecules in nanopores could facilitate a widerange of structure-function studies of membrane proteins by solid-state NMR in the native-like environment. Fully hydrated membrane proteins could be exposed, for example, tobuffers at various pH, ion and drug concentrations. Furthermore, we show that as little asa single lipid bilayer can be stabilized inside the nanopore and diameter of this nanoporecould be varied from ca. 10 to 200 nm providing a unique opportunity to study such struc-tures by high resolution solid-state NMR.Chekmenev, E. Y.; Hu, J.; Gor’kov, P. L.; Brey, W. W.; Cross, T. A.; Ruuge, A. and Smirnov,A. I. 2005. 15N and 31P solid-state NMR study of transmembrane domain alignment ofM2 protein of influenza A virus in hydrated cylindrical lipid bilayers confined to anodicaluminum oxide nanopores. J. Magn. Reson. accepted. Author Disclosure Block: E.Y.Chekmenev, None; J. Hu, None; P.L. Gor’kov, None; W.W. Brey, None; T.A. Cross, None;A. Ruuge, None; A.I. Smirnov, None.

POS-L24 BOARD #LB24NMR Studies of α-synuclein, a Parkinson’s disease associated protein. Yoon-HuiSung1, Robert Bussell, Jr2, David Eliezer, Ph.D1. 1Department of Biochemistry andProgram in Structural Biology, Weill Medical College of Cornell University, New York,NY, USA, 2Department of Physiology, Biophysics and Molecular Medicine, WeillMedical College of Cornell University, New York, NY, USA.α-synuclein (αS) is a synaptic protein that is linked to early onset hereditary forms ofParkinson’s disease (PD) and forms amyloid fibrils that appear in intraneuronal Lewy bodydeposits in the brains of PD victims. We have used high resolution solution state NMRspectroscopy to characterize the structural properties of wild type αS and its two disease-linked mutants, A30P and A53T, bound to lipid mimetic detergent micelles. We find thatthe lipid bound form of αS is partitioned into lipid-associated and lipid free regions andthat the lipid-associated region adopts a highly extended helical structure with a singlebreak. Furthermore, we show that the lipid-associated helical structure adopts an unusualperiodicity, completing three full turns every 11 residues, as a consequence of the 11-mertandem repeats found in the primary sequence of the protein. The two disease-linked αSmutations do not significantly perturb this structure, but the A30P mutations reduces theaffinity of the protein for lipid membranes, possibly by disrupting a helix nucleation site weobserve in the free protein. A search of protein sequence databases reveals several other pro-tein families that possess synuclein-like 11-mer repeats and suggests that these repeats mayplay an important functional role in the lipid interactions of these proteins. AuthorDisclosure Block: Y. Sung, None; R. Bussell, None; D. Eliezer, None.

POS-L25 BOARD #LB25Microcalorimetry of EmrE, a Multi-Drug Transporter. David Miller, Paula Booth.Bristol University, Bristol, United Kingdom.The efflux of structurally diverse toxic substrates by multi-drug transporters is the cause ofsignificant resistance of pathogenic bacteria to a number of therapeutic compounds. EmrEis a small multi-drug transporter from Escherichia coli that utilises the proton gradient toextrude a number of hydrophobic cations from the cell, thus resulting in resistance to toxiccompounds. Current evidence suggests EmrE exists as an unusual asymmetric homodimer,but the method of transport is presently uncertain. Protein-ligand interactions can be meas-ured by the associated heat of the interaction by microcalorimetry, which also allows non-invasive determination of absolute thermodynamic quantities. Interactions can also bemeasured and quantified in lipid envirmonments that are not amenable to other biophys-ical techniques. We have investigated the thermodynamic parameters of binding of the sub-strate tetraphenylphosphonium (TPP+) to EmrE, reconstituted in various detergent andlipid environments, by Isothermal Titration Calorimetry. The Stoiciometry and bindingaffinity is in agreement with previous work, with a stoichiometry of 1 molecule of ligandto each EmrE dimer. Author Disclosure Block: D. Miller, None; P. Booth, None.

POS-L26 BOARD #LB263D Structure and Dynamics of Phospholamban as seen by multi-dimensional magic-angle-spinning NMR. Stefan Becker1, Ovidiu C. Andronesi1, Karsten Seidel1, HowardS. Young2, Marc Baldus1. 1Max Planck Institute for Biophysical Chemistry, Dept. forNMR based Structural Biology, Goettingen, Germany, 2University of Alberta, Dept. ofBiochemistry, Edmonton, AB, Canada.Phospholamban (PLN) is a 52 residue membrane protein, that regulates cardiac contrac-tility. It modulates the Ca-ATPase (SERCA), which mediates the active transport of calci-um into the lumen of the sarcoplasmic reticulum (SR). According to solution-state NMR(1-3), the monomeric mutant (AFA-PLN) comprises an alpha-helical cytoplasmic domainand a single transmembrane helix that are connected by a semi-flexible hinge region. Onthe other hand, mutagenesis studies and recent EPR measurements (4) imply, that strongdynamics in the cytoplasmic portion of PLN play an important role in the functional inter-action with the Ca-ATPase. We have designed a novel set of multi-dimensional solid-stateNMR experiments that permit probing the structure and dynamics of uniformly[13C,15N] labeled phospholamban (PLN) in lipid bilayers. Results obtained on AFA-PLNin the absence and presence of the SERCA1 isoform of Ca-ATPase in lipid bilayers areshown.References:1. Pollesello, P., Annila, A., and Ovaska, M. (1999) Biophys. J. 76, 1784-1795.2. Lamberth, S., Schmid, H., Muenchbach, M., Vorherr, T., Krebs, J., Carafoli, E., andGriesinger, C. (2000) Helvetica Chimica Acta 83, 2141-2152.3. Zamoon, J., Mascioni, A., Thomas, D. D., and Veglia, G. (2003) Biophys. J. 85, 2589-2598.4. Karim, C. B., Kirby, T. L., Zhang, Z. W., Nesmelov, Y., and Thomas, D. D. (2004) Proc.Natl. Acad. Sci. U. S. A. 101, 14437-14442.Author Disclosure Block: S. Becker, None; O.C. Andronesi, None; K. Seidel, None; H.S.Young, None; M. Baldus, None.

POS-L24 BOARD #LB27Solid-State NMR Investigations of Molecular Recognition and Biological Function atInterfaces: A Study Featuring Piscidins, Cationic Antimicrobial Peptides from Fish.Myriam Cotten, Yelena N. Nikolayeva, Shiela M. Jones, Lorraine M. Homem, Tim J.Wagner, McKenna N. Manion, Ken C. Daugherty, Ann J. Auman, Mary J. Ellard-Ivey.Pacific Lutheran University, Tacoma, WA, USA.This research features the principles underlying molecular recognition and biological func-tion of membrane-interacting amphipathic cationic antimicrobial peptides (ACAPs). Theirinitial targets appear to be negatively-charged microbial membranes. Factors that may beimportant for their interactions with biological membranes include their amphipathicstructures, molecular volumes, and aggregation states. Therefore characterizing their sec-ondary structures at high resolution will help establish relationships between their structur-al motifs, interactions with surfaces, activities, and mechanisms of action.Our efforts have been focused on piscidins from fish immune cells. They are not only thefirst ACAPs ever found in the mast cells of animals but they are also believed to play a cru-cial, direct role in the fight against many infections. Peculiar features of the three 22-merpiscidins include: 1) An amidated form, 2) Tolerance to high salt concentrations; 3) Highlyconserved amino ends; 4) Highly cationic; 4) High content of His.Our approach includes a combination of solid-state NMR techniques to probe local struc-ture and dynamics, and the precise nature of peptide-lipid interactions under changing con-ditions. First, we have obtained the 13C CPMAS carbonyl spectra of site-specific isotopi-cally-labeled piscidin-1 in the membrane-bound and unbound states. Secondly,13C1i/15Ni+4 distances measured at low temperature using REDOR have been used to char-acterize the peptide secondary structure in hydrated lipid bilayers. Third, to complementthis data, we are initiating the study of oriented samples. Circular Dichroism studies havealso been performed to assess the global structure of the peptide under sample conditionsmirroring the ones used for the NMR studies. Finally, we have investigated the antimicro-bial activity of these peptides on gram-+/- bacteria. The results of these studies could be sig-nificant in understanding these peptides’ mechanism of action. Author Disclosure Block:M. Cotten, None; Y.N. Nikolayeva, None; S.M. Jones, None; L.M. Homem, None; T.J.Wagner, None; M.N. Manion, None; K.C. Daugherty, None; A.J. Auman, None; M.J.Ellard-Ivey, None.

POS-L28 BOARD #LB28The Hydrophobic Mismatch of Lactose Permease Helix 1. Michael Bennett1, VincentLemaître2,3, Maurits de Planque2, Anthony Watts2, Philip Yeagle1. 1University of Connecticut, Storrs, CT, USA, 2University of Oxford, Oxford, UnitedKingdom, 3Nestec S.A, Vers-Chez-Les-Blanc, Switzerland.The first helix (H1) in the crystal structure of lactose permease is an alpha helix with a slightbend. Solution NMR of a peptide corresponding to H1 demonstrates that the stability ofthis conformation is independent of tertiary contacts from the intact molecule. Thehydrophobic surface of this helix exceeds the hydrophobic thickness of a DMPC bilayer.Solid State NMR experiments have shown that a synthetic peptide corresponding to H1introduces disorder in the packing of DMPC bilayers, revealing that the hydrophobic mis-match is not satisfied by thickening the bilayer. We carried out Molecular Dynamics sim-ulations to investigate the hydrophobic mismatch of H1 in POPE. The MD simulationshave shown that rather than tilting the entire helix to fit into the bilayer, the hinge of H1flexes to accommodate the shorter hydrophobic thickness of POPE. Author DisclosureBlock: M. Bennett, None; V. Lemaître, None; M. de Planque, None; A. Watts, None; P.Yeagle, None.

POS-L29 BOARD #LB29The mobility of plasma membrane associated proteins analysed by FRAP. Farzana K.Bhatti, Pharmacology1, David Brough2, Robin F. Irvine1. 1University of Cambridge, Cambridge, United Kingdom, 2University of Manchester,Manchester, United Kingdom.GAP1IP4BP and GAP1m are members of the GAP1 family, and both interact specificallywith inositol lipids. However, whereas Gap1m is a cytosolic protein that translocates to the

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plasma membrane in response to PtdIns(3,4,5)P3 generation, GAP1IP4BP is constituitivelybound to the plasma membrane in resting cells, a localisation suggested to be mediated byits interaction with PtdIns(4,5)P2 (Cozier et al. 2000, J Biol Chem .). We have explored byfluorescence-recovery after photobleaching (FRAP) the mobility (that is, the diffusion coef-ficient and the fraction of protein that is mobile) of GFP-tagged GAP1IP4BP and GAP1m

transfected into HEK-293 cells. These parameters have been compared when the GAP1sare either cytosolic or in the plasma membrane. Also, we have in turn compared them withthe mobilities of two other plasma membrane-localised proteins: another PtdIns(4,5)P2-binding protein, the PH-domain of PI-PLCδ1, and ICAM, a single transmembrane-span-ning domain protein. The data suggest that when proteins associate with inositol lipids inthe plasma membrane they retain a high mobility, suggesting a free diffusion within theplane of the membrane, which is similar whether they are bound to PtdIns(3,4,5)P3 orPtdIns(4,5)P2. Moreover, the association of GAP1IP4BP is not just mediated byPtdIns(4,5)P2 (Cozier et al), but involves an interaction with other cellular components,probably the sub-membrane skeleton. The mobility of GAP1IP4BP is not detectably alteredby generation of Ins(1,3,4,5)P4 in the cell. Author Disclosure Block: F.K. Bhatti, None; D.Brough, None; R.F. Irvine, None.

POS-L30 BOARD #LB30Measurement of the lateral diffusion of CFTR on the cell surface using an engineeredbiotinylation target sequence. Ian R. Bates, Yishan Luo, Jie Liao, Alexia Bachir, DavidL. Kolin, Ben Hebert, Paul W. Wiseman, John W. Hanrahan. McGill University,Montreal, PQ, Canada.The membrane mobility and protein interactions of the cystic fibrosis transmembrane con-ductance regulator (CFTR) was studied by engineering a biotinylation target sequence intothe 4th extracellular loop and stably expressing the construct in baby hamster kidney (BHK)cells. The target sequence in CFTR was enzymatically biotinylated using biotin ligase, andcould then be labeled with a streptavidin conjugated fluorophore. Inserting the sequencehad little effect on protein expression according to Western blots, or channel activity asassessed by iodide efflux and patch clamp assays. Confocal microscopy of cells exposed tofluorophore-conjugated streptavidin revealed strong plasma membrane staining of cellsexpressing biotinylatable wild-type CFTR, but not those expressing a tagged version of thedisease-associated mutant delta-F508. The mobility of CFTR was studied using fluores-cence recovery after photobleaching (FRAP), using image correlation spectroscopy (ICS).The FRAP recovery curves are consistent with a randomly diffusing population with a sig-nificant immobile population (~20%). The effective diffusion coefficient using FRAP wascalculated through simulated diffusive recovery into a strip bleach, with a D of 1.4-2.1 x10-10 cm2sec-1. The ICS method resulted in a diffusion coefficient of 3.6-7.8 x 10-11 cm2sec-

1, approximately 4 times slower than FRAP. Computer simulations indicated that a con-fined, slowly moving population would result in a faster diffusion using FRAP. To investi-gate possible protein interactions that could result in this immobile pool, fluorophoretagged CFTR was co-expressed with a Clathrin-GFP and glycosylphosphoinositol (GPI)-GFP. CFTR was found to co-localize with Clathrin on the cell surface, but not with GPI-GFP suggesting that one of the interactions that results in the immobile population couldbe the result of Clathrin pit formation, and not to lipid rafts. Author Disclosure Block: I.R.Bates, None; Y. Luo, None; J. Liao, None; A. Bachir, None; D.L. Kolin, None; B. Hebert,None; P.W. Wiseman, None; J.W. Hanrahan, None.

POS-L31 BOARD #LB31Proton and Hydroxide Migration in Aquaporins. Morten Ø. Jensen1, Carme Rovira2.1University of Southern Denmark, Odense, Denmark, 2Centre de Recerca en QuímicaTeòrica, Parc Científic de Barcelona, Barcelona, Spain.Non-equilibrium transfers of a hydroxide ion and of a proton along the linear water chainin Aqua(glycero)porin GlpF from Esherichia Coli are studied by ab initio Car-Parrinellomolecular dynamics simulations within a QM/MM approach.It is found that the protein(MM treated)stabilizes a bipolar single-file water configuration (QM treated). The single-file chain features a contiguous set of water-water hydrogen bonds in which polarization ofthe water molecules vary strongly with position along the channel axis.De-protonation ofthe water chain alters its hydrogen bonding properties pruning water molecules within thechain to reorient while the hydroxide ion migrates via proton hops of the neighboring watermolecules in less than one picosecond.Counter-intuitively,the hydroxide ion is not attract-ed by a positively charged,conserved,channel lining, arginine residue (R206)but immobi-lized at two centrally located,conserved asparagine-proline-alanine (NPA)motifs. A fourfoldcoordination of the hydroxide ion at the channel center,similar to the one found in ab ini-tio MD simulations of an hydroxide ion in solution,stabilizes the hydroxide ion in this posi-tion.Elimination of electrostatic interactions with the two N side-chains of the NPA motifsincreases the escape rate of the trapped hydroxide ion.Protonation also alters hydrogenbonding properties of the wire, in turn facilitating water reorientation and subsequent pro-ton expelling.In the periplasmic half of the water wire,expelling occurs via the Grothussmechanism.Protonation in the cytoplasmic half implies breakage of the water wire at theNPA motifs where ion-channel electrostatic repulsion is maximal and water-channel elec-trostatic interactions are minimal.In this region,the proton, essentially solvated as an Eigen-complex is diffusively rejected. Author Disclosure Block: M.Ø. jensen, None; C. Rovira,None.

POS-L32 BOARD #LB32Functioning of the P-glycoprotein Multidrug Efflux Pump: a New Integrated KineticModel. Frances J. Sharom1, Miguel R. Lugo2. 1University of Guelph, Guelph, ON,Canada, 2Universidad Central de Venezuela, Caracas, Venezuela.P-glycoprotein (Pgp) is an ABC transporter that couples ATP hydrolysis to export of a vari-ety of hydrophobic compounds. To date, kinetic models of Pgp functioning have either (1)considered the passive flux of substrate through the membrane and reduced the catalyticcycle to a simple function of pumping, with VM and Km as kinetic parameters for trans-porter-mediated efflux, or (2) constructed a detailed catalytic cycle of the protein out of thecontext of the membrane and passive fluxes. Here we present an integrated model that com-bines both approaches. Our model consists of four physical compartments (internal medi-um, inner leaflet, outer leaflet and external medium) and a chemical entity, Pgp. Substrate

molecules are distributed between each compartment according to their partition coeffi-cient, and the fluxes are described by absolute reaction rates. Pgp is envisaged as a shortcut(with variable effective permeability) between the inner leaflet of the bilayer and the exter-nal medium (a hydrophobic vacuum cleaner) which alters substrate partitioning amongcompartments. For the catalytic cycle of Pgp, our scheme is based on the single carriermodel, but with both forward transition rate constants (for the loaded and empty carrier)dependent on ATP. This integrated model is compatible with the biochemistry of PgpATPase activity in solution and membranes, as well as transport activity in proteoliposomesand intact cells. The model can explain: (i) basal ATPase activity; (ii) activation, inhibitionor biphasic drug-dependence of ATPase activity; (iii) saturable ATP concentration depend-ence of basal and total ATPase activity; (iv) similar affinities of Pgp for substrate loading andunloading (v) coupling of transport to ATP hydrolysis; (vi) generation of a substrate con-centration gradient. Author Disclosure Block: F.J. Sharom, None; M.R. Lugo, None.

POS-L33 BOARD #LB33Light induced and thermal conversions between Rhodopsin intermediates. Franz J.Bartl, Eglof Ritter, Kerstin Zimmermann, Bernhard Knierim, Oliver P. Ernst, KlausPeter Hofmann, Martin Heck. Institut f. Medizin. Physik und Biophysik, Berlin,Germany.Rhodopsin is the only protein of the visual cascade that directly interacts with light. In itsdormant ground state the chromophore 11-cis- retinal is bound to the apoprotein opsin viaa protonated Schiff base linkage to Lys296. Upon light absorption the retinal changes itsgeometry to all-trans (first switch). The receptor proceeds through a number of intermedi-ates leading to the active Meta II state which features a deprotonated Schiff base and existsin a pH- and temperature-dependent equilibrium with its precurserprecursor Meta I con-taining a deprotonated Schiff base. Blue light absorption of Meta II does not lead back tothe ground state by trans/cis isomerization of the polyene chain, but to Meta III via syn/antiisomerization of the Schiff base (second switch).We find that light induced Meta III formation is only observed when light is applied toMeta II. Light induced Meta III formation is not found with any other intermediates, con-taining a protonated Schiff base including Meta I. We propose that in the active state, theprotein environment exerts a constrain on the poleyene chain of the chromophore, thuspreventing central double bond trans/cis isomerization. Reaction schemes for the lightinduced and thermal conversions between the rhodopsin intermediates are presented.Author Disclosure Block: F. Bartl, None; E. Ritter, None; K. Zimmermann, None; B.Knierim, None; O. Ernst, None; K. Hofmann, None; M. Heck, None.

POS-L34 BOARD #LB34Crystal Structure of the Yeast Nicotinic Acid Phosphoribosyltransferase (NAPRTase) at1.75Å Resolution. Joshua S. Chappie, MS1,2, Jaume M. Canaves, PhD3,2, Raymond C.Stevens, PhD1,2. 1The Scripps Research Institute, La Jolla, CA, USA, 2Joint Center forStructural Genomics, La Jolla, CA, USA,3.The San Diego Supercomputer Center, La Jolla, CA, USA.Metabolic salvage pathwaysallow organisms to replenish vital cofactors from existing intermediates rather than synthe-size them de novo. One such recovery route allows the recycling of nicotinamide adeninedinucleotide (NAD) from its degradation product nicotinamide in a limited number ofchemical steps. To fully understand the biological relationships underlying these specificpathways, it is necessary to decipher the structural and functional details of their enzymat-ic components. Using the Joint Center for Structural Genomics (JCSG) high-throughputstructural genomics pipeline, we solved the structure of the yeast nicotinic acid phosphori-bosyltransferase (NAPRTase), the second key enzyme in the NAD salvage pathway, at1.75Å resolution. We find a two-domain architecture that resembles the design of quino-linic acid phosphoribosyltransferases (QAPRTases), a related family of enzymes involved inde novo NAD production. Further structural comparison reveals that these segments occu-py different orientations relative to one another in each case, permitting the NAPRTase toexist as an open monomer while the QAPRTases assume a head-to-tail dimeric arrange-ment. This modular design, together with computational modeling and bioinformaticsdata, implies a general mechanism for understanding the evolution of NAD biosyntheticpathways and the structural relationships contained therein. Author Disclosure Block: J.S.Chappie, None; J.M. Canaves, None; R.C. Stevens, None.

POS-L35 BOARD #LB35Structure of the FAD-based Redox Sensor Domain of NifL at Atomic Resolution:Insights Into PAS Domain Dimerization, Mechanism, and Signaling. Jason M. Key1,Marco Hefti2, Erin Purcell1, Keith Moffat1. 1University of Chicago, Chicago, IL, USA,2Wageningen University, Wageningen, The Netherlands.NifL is a multi-domain sensor protein responsible for the detection of oxygen and ADP inthe free-living diazotroph Azotobacter vinelandii. The site for oxygen recognition within thisprotein is an FAD-based PAS domain that is similar to FMN-based photoreceptors. Thisdomain detects oxygen via a change in the redox state of its FAD cofactor. In order toexplore the mechanism of redox mediation signal recognition and transduction in this pro-tein, we have determined the crystal structure of the N-terminal FAD-bound PAS domain,AvNifLF, to atomic resolution (1.04 Å). The structure of this protein reveals a conserveddimer of two PAS domains which are very similar to the structures of EcDosH andSmFixLH reported previously. The AvNifLF protein contains a novel cavity within the coreof the PAS domain which contains two bound water molecules coordinated in direct prox-imity to the FAD prosthetic group. Using X-ray photoreduction of the FAD group, wevisualize the initial conversion events from the oxidized “off” state of the protein toward areduced “on” state. This reveals the formation of a redox-sensitive hydrogen bond betweenthe side chain of glu70 and ser39 and rearrangement of the water molecules and polar sidechains within the FAD cavity. We propose that these are the initial events in the conversionbetween signaling states within this bacterial redox sensor. Structural differences propagatefrom the redox switch and are visible 7Å distant, on the residues of the A and I strands ofthe beta-sheet of the protein in a region implicated in signaling in other PAS domain pro-teins through changes in quaternary structure. Author Disclosure Block: J.M. Key, None;M. Hefti, None; E. Purcell, None; K. Moffat, None.

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POS-L36 BOARD #LB36INHOMOGENEITY OF TYPE I COLLAGEN GELS. Olga S. Latinovic, M.S., H.Daniel Ou-Yang, Ph.D. Lehigh University, Bethlehem, PA, USA.This paper reports a study of inhomogeneity of the gels by comparing the structural andmicromechanical properties of Type I Collagen gels. Based on forced oscillations and ther-mal motions of the particles embedded in the gel matrix, and using oscillating optical tweez-ers, we are able to determine local viscoelastic response of the matrix in the vicinity of theprobe particle. Phase contrast and confocal fluorescence microscopy studies reveal phaseseparation in the gels with sparse and dense regions as a consequence of randomly inter-connected fibers into brunched networks. The inhomogeneity persists through the sol-geltransition. This study establishes correlation between the structural inhomogeneity andmicromechanical properties in terms of local network density. Author Disclosure Block: O.Latinovic, None; H. Ou-Yang, None.

POS-L37 BOARD #LB37Using Networks to Identify Functionally Relevant Differences Between Similar ProteinStructures. Liskin Swint-Kruse. KU Medical Center, Kansas City, KS, USA.The field of structural proteomics faces a new challenge in determining which fine struc-tural details endow unique in vivo functions to sequences that assume a common fold.Problems of similar detail are also encountered when exploring how distinct conformationscontribute to different states of an individual protein’s dynamic function. However, thecomplexity of these large, three-dimensional molecules hinders direct visual comparisons,while C? r.m.s.d. calculations lose important information from side chains. In networkanalysis, structural data are re-cast in a two-dimensional format that includes side chaininformation. These diagrams have been useful for contrasting multiple structures, includ-ing (1) interfaces of homologous proteins, (2) structural time points throughout a dynam-ics simulation, and (3) functionally different conformations of a single protein. All examplecomparisons have resulted in new hypotheses to test experimentally. A computer programnamed Resmap is being created to automate this process. This program will be available todownload to computers running Windows PC platforms and will allow users to defineatom types and bond lengths (e.g. the types and distance between atoms of a hydrogenbond). Author Disclosure Block: L. Swint-Kruse, None.

POS-L38 BOARD #LB38Structural Alignment Methods VAST and SHEBA Evaluated Against SCOP. VichetraSam1, Chin-Hsien Tai2, Jean Garnier1, Jean-Francois Gibrat3, Byungkook Lee2, Peter J.Munson1. 1MSCL/DCB/CIT/NIH/DHHS, Bethesda, MD, USA,2LMB/CCR/NCI/NIH/DHHS, Bethesda, MD, USA, 3MIG/INRA, Jouy-en-Josas,France.Many protein structural alignment methods have been developed and used for the classifi-cation of protein domains. However, different methods give rise to significantly differentresults. These differences affect the protein domain classification and our view of the pro-tein structure universe.In order to study the kind of differences that result from using different methods and togain insight into the mechanism by which these differences arise, we conducted an in depthcomparison of VAST (Current Opin Struct Biol, 1996;6:377-385), SHEBA (Protein Eng2000;13:233:123-138) and SCOP (J. Mol. Biol. 1995, 247, 536-540). SCOP is a manu-ally curated protein domain structural classification database, often considered as the goldstandard of protein structure classification. VAST achieves alignment by clustering vectorsthat represent secondary structural elements, and is used by NCBI/Entrez. SHEBA struc-ture comparison uses sequence homology and the structural profile for each residue toobtain the initial alignment.First, we have developed a statistical method for evaluating two structure comparison meth-ods against SCOP. Comparison of SHEBA and VAST reveals their respective weaknessesand possible ways for future improvement. The comparison with SCOP shows that bothSHEBA and VAST agree well with SCOP.Important fold assignment confusions have also been noted: Some domain pairs thatbelong to the same SCOP fold are considered to be structurally dissimilar by SHEBA orVAST. Conversely, some domain pairs considered to be similar by SHEBA or VAST belongto two different SCOP folds. These discrepancies between automatic comparisons and amanual curation are quantified. Some discrepancies may arise because automated methodsseem to look for uniform, global geometric similarity while the manual method mayemphasize relevant local similarity and perhaps other functional and biological factors.Author Disclosure Block: V. Sam, None; C. Tai, None; J. Garnier, None; J. Gibrat, None; B.Lee, None; P.J. Munson, None.

POS-L39 BOARD #LB39NMR solution structure and backbone dynamics of the tick-borne Langat flavivirus E-protein domain III suggests a specific viral epitope. Munia Mukherjee, Ph.D.1,Kaushik Dutta, Ph.D.2, Mark A. White, Ph.D.1, David Cowburn, Ph.D.2, Robert O.Fox, Ph.D.1. 1University of Texas Medical Branch, Galveston, TX, USA, 2New YorkStructural Biology Center, New York, NY, USA.Flaviviruses cause many human diseases including dengue fever, yellow fever, West Nilevirus encephalitis, and hemorrhagic fevers; and are transmitted to their vertebrate hosts byinfected mosquitoes and ticks. Domain III of the envelope protein (E-D3) is considered tobe the primary viral determinant involved in the virus-host cell receptor interaction, andthus represents an excellent target for an antiviral drug development. Langat (LGT) is a nat-urally attenuated BSL-2 TBE virus and is a model for the pathogenic BSL-3 and BSL-4viruses in the serogroup. We have determined the solution structure of LGT-E-D3 usingheteronuclear NMR spectroscopy. The backbone dynamics of LGT-E-D3 has been inves-tigated using 15N relaxation measurements. A detailed analysis of the solution structureand dynamics of LGT-E-D3 suggests potential residues that could form the surface of theviral epitope for antibody recognition and thereby represents a target site for antiviral ther-apeutic design. Author Disclosure Block: M. Mukherjee, None; K. Dutta, None; M.A.White, None; D. Cowburn, None; R.O. Fox, None.

POS-L40 BOARD #LB40Structural motifs for the self-assembly of lanreotide into nanotubes. Anjali Pandit,PhD1, Nadege Jamin, PhD1, Celine Valery, PhD2, Roland Cherif-Sheikh, PhD2, MaitePaternostre, PhD1. 1CEA Saclay, Gif sur Yvette, France, 2Ipsen Pharma S.A., Barcelona,Spain.The synthetic octapeptide lanreotide (DNaph-Cys-Tyr-DTrp-Lys-Val-Cys-Thr-CONH2)has exceptional self-organizing properties. Lanreotide is a growth hormone inhibitor andhas been designed by the pharmaceutical company Beaufour-Ipsen for treatment ofagromegaly. In the treatment, a high concentration of lanreotide is mixed with water, spon-taneously forming a hydrogel that is implanted under the skin. The specific gel-formingproperties of lanreotide permit a month-long continuous release of peptides in the blood-stream, in contrast to an average lifetime of several minutes observed for single peptides.Structural studies showed that this gel consists of unique three-dimensional structures, inwhich peptides are self-assembled into rigorous monodisperse hollow nanotubes of 24 nmdiameters. The nanotubes are built up from curled filaments, consisting of anti-parallelstacked hairpin-folded peptides. At low peptide concentrations, these tubes are arranged ina hexagonal network and at very high peptide concentrations nanotubes embedded in othernanotubes appear.In this work we search for the structural motifs that are responsible for this special form ofself-organization. Different spectroscopic techniques are combined to measure the confor-mations of lanreotide in monomers, filaments and nanotubes. The importance of the aro-matic residues, net charge and the disulfide bridge for self-assembly are probed by selectivemodification of the respective groups. Results reveal the role of the naphthyl-alanine in driv-ing the self-association process. We aim to specify intra- and inter-peptide interactions thatmay direct the formations and shapes of the observed assembled structures.Key words: octapeptide, nanotubes, aromatic residues, NMR, fluorescence, Raman spec-troscopy. Author Disclosure Block: A. Pandit, None; N. Jamin, None; C. Valery, IpsenPharma S.A. Employment (full or part-time); R. Cherif-Sheikh, Ipsen Pharma S.A.Employment (full or part-time); M. Paternostre, None.

POS-L41 BOARD #LB41Solubility Studies of Ribonuclease Sa. Saul R. Trevino, B.S., J. Martin Scholtz, Ph.D.,C. Nick Pace, Ph.D. Texas A&M University, College Station, TX, USA.Increasing protein solubility is of interest in several areas of research. The goal of this studyis to identify mutations that significantly increase the solubility of Ribonuclease (RNase) Sa.Rational mutagenesis guided by structure and homology has been used to assess the con-tributions of surface polarity and net charge to protein solubility. For example, a multiplesequence alignment of RNase Sa and four other microbial ribonucleases was used to iden-tify homologous hydrophobic to polar mutations in loop regions. The correspondinghydrophobic to charged mutations have also been made to determine the impact of netcharge on solubility. Homologous mutations were used to minimize the perturbation of thestructure. We hypothesize that hydrophobic to polar (or charged) mutations produce moredramatic increases in solubility if the mutated position is flexible. Therefore, an additionalcriterion in the design of these mutants is high flexibility of the substituted positions asdetermined by H/D exchange data. Results and discussion of this study are forthcomingand will be presented. Author Disclosure Block: S.R. Trevino, None; J.M. Scholtz, None;C.N. Pace, None.

POS-L42 BOARD #LB42A Functional Monomeric Plague Vaccine Created by Circular Permutation. David A.Chalton1, Julie A. Musson1, Nichola Walker2, Alistair McGregor1, E. DianeWilliamson2, Julie Miller2, John H. Robinson1, Jeremy H. Lakey1. 1University of Newcastle Upon Tyne, Newcastle, United Kingdom, 2Dstl, Salisbury,United Kingdom.Yersinia pestis is the causative agent of plague, a zoonotic infection that usually passes froma natural reservoir in rodents to humans via a bite from an infected flea Xenopsylla cheopis.Capsular antigen Fraction 1 (F1) from Y. pestis is produced during growth above 33oC,and serves to act as a protective structure around the bacteria and confers antiphagocyticproperties on it. Y. pestis lacking F1 show an increased susceptibility to phagocytosis bymacrophages, though not necessarily a reduction in virulence.F1 forms large aggregates which makes structure-function studies problematic. The X-raystructure has been determined as a complex with its chaperone Caf1M, and revealed thatthe polymeric nature of the protein results from a domain swap of an N terminal β-strandinto a cleft of the adjoining monomer.Examination of the published structure and protein engineering has allowed us to constructa monomeric, folded form of F1. A connecting linker and C-terminal extension consistingof the F1 N-terminal region, allowed the generation of a protein which is able to self stab-lise (i.e. is circularly permuted), and thus forms a cooperatively folded, thermally stablemonomeric protein.Here we present data from the monomeric circularly permuted form of F1, which possess-es near wild type characteristics. We have analysed this novel protein, by a number of bio-physical methods; circular dichroism, thermal stability assays, differential scanningcalorimetery, and intrinsic fluorescence; as well presenting protease protection data, T-cellproliferation assays and immunogenicity data. Author Disclosure Block: D.A. Chalton,None; J.A. Musson, None; N. Walker, None; A. McGregor, None; E. Williamson, None; J.Miller, None; J.H. Robinson, None; J.H. Lakey, None.

POS-L43 BOARD #LB43Repair Mechanisms of (6-4) Photolesions in DNA by (6-4) Photolyase. ChristopherHarrison, Olaf Wiest. University of Notre Dame, Notre Dame, IN, USA.Ultraviolet irradiation of DNA induces formation of the (6-4) photolesion, 1, damaginggenetic information, leading ultimately to skin cancer. Through an unknown mechanism,1 may be repaired in vivo by (6-4) photolyase. Due to the instability of an oxetane inter-mediate, 2, and the absence of an x-ray structure of (6-4) photolyase, the currently proposedmechanism (Figure 1) cannot be validated. With an endothermic difference of 15.5kcal/mol from 1 to 2, it is difficult to envision enzymatic thermal formation and stabiliza-

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tion of 2 from 1 followed by latent ET to yield 2·- and ultimately two thymine monomers,3. Alternative repair mechanisms are investigated using ab initio calculations.Docking of 1 to a (6-4) photolyase sequence homology model provided a model enzyme-substrate complex, followed by 5ns MD simulations. Catalytically relevant histidines foundin contact with 1 implicate their possible role as hydrogen donors/acceptors to yield 3.Further investigations of the enzyme’s contribution to catalysis will be investigated via con-tinued molecular dynamics simulations.

These studies, utilizing molecular dynamic simulations, coupled with ab initio investiga-tions, will provide insights towards understanding the skin cancer protection mechanismsof (6-4) photolyases. Author Disclosure Block: C. Harrison, None; O. Wiest, None.

POS-L44 BOARD #LB44Structure and Dynamics of the T7 DNA Polymerase-DNA Complex in the Presenceof Aminofluorene-Guanosine and Acetylaminofluorene-Guanosine in the TemplateStrand of the Bound DNA. Lance R. Pickens, Jan Florian. Loyola University of Chicago, Chicago, IL, USA.Carcinogenic DNA adducts such as acetylaminofluorene (AAF) and aminofluorene (AF)

cause mutations during DNA synthesis in Escherichia coli. While havingonly minor structural differences, these compounds exhibit major func-tional differences both in vivo and in vitro. The T7 phage polymerasecrystal structure with either adduct in the pre-insertion position werestudied by molecular dynamics calculations. The role of AF-dG andAAF-dG lesions in mutagenesis was further investigated by examiningthe dynamics of the lesions in the templating position, and by examiningfree energies for binding of correct (dCTP) and incorrect (dATP, dTTP)dNTP substrates opposite dG, AF-dG and AAF-dG templating bases.Our simulations revealed the AF-dG adduct is capable of a greater degreeof conformational change than its AAF-dG counterpart and that thelarge conformational change required for the transfer of the base frompre-insertion to insertion position is significantly hindered with the AAF-dG adduct. This may explain, in part, the reason why the AAF-dG basecauses frameshifts in vivo while AF-dG bases, which are more conforma-

tionally free, tend to induce substitution mutations. Author Disclosure Block: L.R. Pickens,None; J. Florian, None.

POS-L45 BOARD #LB45Simple Rules Govern the Dynamic Properties of a Transcriptional Switch.Dorothy Beckett, PhD, Emily D. Streaker. Univ Maryland, College Park, MD, USA.The response of an organism to environmental and metabolic cues requires communica-tion between transcription regulatory processes and other cellular events. A number of bio-logical control circuits have been identified in which this communication is achieved by asingle multi-functional protein that participates both directly in transcription initiation aswell as in at least one other process. The dimerization cofactor of hepatocyte nuclear factor(DCoH) and β-catenin are representative of this class of proteins. Results of structural stud-ies suggest that for each of these proteins, function is dictated by the formation of alterna-tive and mutually exclusive protein:protein interactions. However, the rules that governpartner and, thus, functional switching are not known for any of these proteins. In theEscherichia coli biotin regulatory system the bifunctional protein, BirA, both catalyzes post-translational biotin addition to a biotin-dependent carboxylase and binds sequence-specif-ically to DNA to repress transcription initiation at the biotin biosynthetic operon. Previousstructural and modeling studies suggest that BirA function is determined by formation ofalternative homo- and heterodimeric protein:protein interactions. In this work, functionalswitching of the biotin holoenzyme synthetase/biotin repressor is investigated using rapidquench-flow DNaseI footprinting and MALDI ToF mass spectrometry. Results of thesemeasurements indicate that BirA can initially be selectively targeted toward its enzymaticfunction simply by increasing the rate of heterodimerization relative to that of homod-imerization. Subsequent shifting to its DNA binding function occurs as the pool of het-erodimer partner is depleted and homodimerization dominates. This simple control mech-anism may extend to other members of this class of multi-functional transcription factors.Author Disclosure Block: D. Beckett, None; E.D. Streaker, None.POS-L46 BOARD #LB46Pseudourine 32, 39 containing anticodon stem loop tRNAPhe:Watson-Crick or bifur-cated hydrogen bonds? Izabela Tworowska, Ph. D., Edward P. Nikonowicz, Ph.D. RiceUniversity, Houston, TX, USA.In all tRNA crystal structures published in the NDB databases residues 32 and 38 of theanticodon stem loop are linked through a bifurcated hydrogen bond. Crystal structures ofE. coli tRNACys and yeast tRNAAsp contain pseudouridine (ψ) in the position 32. We arefocused on the interactions between base pairs ψ32-A38 and A31-ψ39 in E. coli ACSLPhe (fig.below). The hydrogen bond networks have been determined using heteronuclear NMRspectroscopy. Our NMR results confirmed that the effect of pseudouridine depends on itslocation and presence of the other modifications. Pseudouridine 32 is stabilizing the stemand has little influence on the loop in the ψ32-ACSLPhe. ψ32 and A38 form a Watson-Cricktype of hydrogen bonds. In the double modified i6A37ψ32-ACSLPhe, stabilizing effect of ψ32is partially compensated by the presence of i6A37. The incorporation of pseudouridine atposition 32 and 39 leads primarily to local perturbations of the helix structure and mini-mally alters the overall fold of the loop region of ACSLPhe. Our NMR experiment exclud-ed the presence of bifurcated hydrogen bonds between ψ32 and A38 in the ψ32,39_ACSLPhe.Author Disclosure Block: I. Tworowska, Rice University Employment (full or part-time); E.P.Nikonowicz, Rice University Research grants, Employment (full or part-time).

POS-L47 BOARD #LB47Structural heterogeneity in DNA. Temperature dependence of time-resolved 2-aminop-urine fluorescence in dinucleotides. Herbert van Amerongen, Prof., Linda Keukens,Arie van Hoek, Oscar J. G. Somsen. Wageningen University, Wageningen, TheNetherlands.2-Aminopurine is a fluorescent base analog that probes local dynamics in DNA because itsfluorescence is quenched by the surrounding bases. But, the underlying quenching mech-anisms are still under investigation. We have studied fluorescence decay of 2-aminopurinein dinucleotides and found a similar heterogeneity as was previously observed in largeroligonucleotides. This indicates that dinucleotides can function as a model for local dynam-ics of intact DNA. It is found that fast (21-35 picosecond) quenching can be induced byall natural DNA bases. The results confirm that the product of this fast transition has thecharacteristics of a dark state. We also observe a strong temperature dependence for the flu-orescence decay profiles. The results indicate that two decay components are due to theexcited-state dynamics of a single conformational state. Our results resemble the gatingmodel, but indicate that only one conformation is initially excited. Author Disclosure Block:H. van Amerongen, None; L. Keukens, None; A. van Hoek, None; O.J.G. Somsen, None.POS-L48 BOARD #LB48Time resolved study of Tetrahymena ribozyme compaction measured with small anglex-ray scattering. Jessica Lamb, Lisa Kwok, Hye Yoon Park, Kurt Andresen, HeatherSmith, Lois Pollack. Cornell University, Ithaca, NY, USA.Using a microfabricated fast laminar flow mixing device in conjunction with small angle x-ray scattering (SAXS), we have monitored global conformational changes during the fold-ing of the Tetrahymena ribozyme under varying conditions. Previous time-resolved SAXSexperiments show that compaction occurs through two kinetic phases on millisecond timescales (R. Russell et al., PNAS 99, p. 4266 2002). We report on recent experiments that testphysical models of riboyzme compaction by comparing the time scale for collapse in wildtype and mutant molecules and under varying solution conditions. Author Disclosure Block:J. Lamb, None; L. Kwok, None; H. Park, None; K. Andresen, None; H. Smith, None; L.Pollack, None.POS-L49 BOARD #LB49Characterization of conformational transition state of Holliday junction via combinedoptical tweezers and single-molecule FRET. Sungchul Hohng, Ph.D., Michelle Nahas,Rahul Roy, Taekjip Ha. University of Illinois at Urbana-Champaign, Urbana, IL, USA.Optical tweezers are the most precise method available for mechanical manipulation of sin-gle-biomolecules while single-molecule FRET is the most widely used techniques for study-ing conformational dynamics of biomolecules. Combining these two techniques wouldprovide an unprecedented opportunity to explore the inner workings of the cellularmachine, allowing researchers to pull biomolecules with optical traps while watching theresponse of such macromolecules through FRET. An immediate application of the com-bined instrument is the characterization of the conformational transition states of biomol-ecules. This is accomplished through the dependence of the forward and backward reactionrates between two states on the relative position of transition state between these states.Here, we report the first realization of optical tweezers and single-molecule FRET and char-acterization of conformational transition state of a DNA four-way junction called the‘Holliday junction’. Two arms of the Holliday junction were tagged with Cy3 (donor) andCy5 (acceptor) to observe conformation changes through FRET while a third arm waslabeled with biotin for surface-immobilization. The remaining arm is tethered to a trappedbead via λ-DNA linker to apply force to a surface-immobilized Holliday junction. Wemeasured conformational transitions using single-molecule FRET at varying applied forces.As the applied force increaseds the, dwell time of the high FRET state became shorter whilethat of low FRET state became longer. This may indicate that the open state is the transi-tion state. By doing dwell time analysis, we expect to get a more complete picture of reac-tion energy landscape of Holliday junction. Author Disclosure Block: S. Hohng, None; M.Nahas, None; R. Roy, None; T. Ha, None.POS-L50 BOARD #LB50The effect of minor groove amino groups and major groove methyl groups on DNAbase-pair dynamics: implications for formation of A-tract structure. SebastianWarmlander, PhD, Mikael Leijon, PhD. Division of Biophysics, Stockholm University,Stockholm, Sweden.In this study base-pair opening dynamics of different RRYY steps in DNA duplexes wereobtained from catalyzed imino proton exchange measured by NMR spectroscopy. Theimportance of a major groove C5-methyl group and a minor groove N2-amino group forthe dynamics was assessed by comparison of AATT, AAUU, GGCC, GGMM, IICC andIIMM steps. We show that both the amino group and the methyl group, separately as wellas together, have a stabilizing effect on the opening dynamics. Interestingly, very similardynamics is observed for the AATT and IIMM steps, where both steps also exhibit theextraordinary opening behaviour of the central base pair that has been associated with anA-tract type of structure (Leroy et al., 1988). Hence, presence of the C5-methyl group andabsence of the N2-amino group seem to be required to form this type of structure.Furthermore, since the monitored imino proton is located at the purine in the IM-pairs,and at the pyrimidine in the AT-pairs, the similarity in opening dynamics implies that theopening event is symmetric with regard to the two bases of the base pair.References: Leroy, J.L., Charretier, E., Kochoyan, M. & Guéron, M. (1988) Biochemistry27, 8894-8898. Author Disclosure Block: S. Warmlander, None; M. Leijon, None.POS-L51 BOARD #LB51Crystal structure of a guanine riboswitch: a basis for ligand specificity by a naturalRNA aptamer. Sunny D. Gilbert, Sarah J. Wise, Crystal Love, Rebecca Montange,Robert T. Batey. University of Colorado Boulder, Boulder, CO, USA.Riboswitches are a level of transcriptional or translational control that is independent of pro-tein cofactors. Molecular recognition of a metabolite is carried out by a regulatory elementin the 5’ UTR of the mRNA molecule leading to an alteration in gene expression. TheseRNA sensors have been discovered for a number of molecules across several species of bac-teria and are known to regulate > 2% of all genes in B. subtilis. In the case of the guanineriboswitch, a loosely associated binding pocket is created to detect the presence of guanineor hypoxanthine to control purine metabolism and synthesis genes. Upon metabolite bind-

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ing, the riboswitch forces the formation of a downstream terminator, halting transcription.We have solved a 1.9 Å resolution crystal structure of the guanine riboswitch bound to themetabolite hypoxanthine. The structure revealed strikingly complex features that con-tributed to the recognition and specificity of binding. The ligand binding pocket com-pletely engulfs the nucleobase, shielding it from solvent. An intricate tertiary loop-loopinteraction, distant from the binding pocket, is also required for metabolite binding.Complimentary studies of binding done by isothermal titration calorimetry (ITC) havemeasured the thermodynamic contribution of each contact to the purine. These data revealthat the specific hydrogen bonds between the ligand and the RNA are principally respon-sible for the high-affinity interaction. Concurrently, we are investigating the binding toler-ance of nucleotides with alternative functional groups and focusing on obtaining structuresof other nucleotides bound to the riboswitch in order to refine our knowledge of the speci-ficity and complexity involved in metabolite recognition and transcription termination.Author Disclosure Block: S.D. Gilbert, None; S.J. Wise, None; C. Love, None; R. Montange,None; R.T. Batey, None.POS-L52 BOARD #LB52Nano-traincars moving down DNA tracks: Single molecule FRET and MD simula-tions of a DNA sliding clamp moving along DNA. Daniel Barsky, PhD1, Ted A.Laurence, PhD1, Christopher W. Hollars, PhD1, Eric J. Yin, PhD1, Julio A. Camarero,PhD1, Michael P. Thelen, PhD1, Kenneth S. Kim, PhD1, Aaron Johnson, PhD2, Mike

O’Donnell, PhD2. 1Lawrence Livermore Nat’l Lab,Livermore, CA, USA, 2Rockefeller Univ and HowardHughes Med Inst, New York, NY, USA.Like nano-traincars-on-tracks, DNA sliding clampproteins can “ride” more than 10,000 bases along aDNA double helix (> 3 ?m linear distance). Theclamps can also be driven along by protein motors andcan carry other proteins along. The clamps accomplishthis by encircling the DNA, a unique topology in biol-ogy. In the absence of other proteins, the DNA slidingclamps apparently “freely slide” along DNA, yet analy-sis of the protein structures, reveals many positivecharges along the inner ring that should create strongsalt bridges with the DNA. Why then do the clampsnot remain stationary on the DNA? And how canclamps proceed past sizeable distortions in the DNA

double helix? By a combined experimental and computational approach, we have investi-gated the interactions between DNA and a DNA sliding clamp protein, the beta subunitof pol III. We have used single-molecule FRET measurements to infer the speed and dif-fusional character of the clamp’s translocation and used MD simulations to illuminate thedetails of the DNA-protein interactions. Work done by US DOE at the University ofCalifornia, Lawrence Livermore National Laboratory. Author Disclosure Block: D. Barsky,None; T.A. Laurence, None; C.W. Hollars, None; E.J. Yin, None; J.A. Camarero, None;M.P. Thelen, None; K.S. Kim, None; A. Johnson , None; M. O’Donnell, None.

POS-L53 BOARD #LB53Two-Photon Standing-Wave Fluorescence Microscopy to Measure Nanometer-ScaleMotions. Christopher J. Bardeen, Sara K. Davis, Ph. D. University of California, Riverside, Riverside, CA, USA.We use a two-photon standing wave fluorescence photobleaching experiment to look at thesmall-scale motions of fluorescently-labeled DNA in a variety of biological systems. In liveXTC-2 and HeLa cells, the motion of the DNA-containing chromatin is constrained andexhibits considerable cell to cell variation. The mean diffusion coefficient in the live XTC-2 cells of 6 x 10-12 cm2/s on the 100 nm lengthscale. In dead cells, and in nuclei isolatedunder physiological conditions, this motion is completely absent. To determine the chem-ical origins of the motion observed in the live cells, we have used photochemical cross-link-ing experiments to covalently link the core histones with the DNA, finding that this stopsall motion. By varying the ionic strength of solutions of isolated nuclei, we are able toreversibly turn the diffusive motion on and off. Both results are consistent with core histonebinding to the DNA playing an important role in allowing the constrained diffusion of thechromatin. Our results suggest that events like nucleosomal sliding on small (10 nm)lengthscales leads to an enhancement of chromatin flexibility on larger (100 nm) length-scales. A newly developed fluorescence correlation variant of the standing-wave experiment,which can be applied to sparsely-labeled samples and which avoids the deleterious effects ofphotobleaching, will also be presented. Author Disclosure Block: C.J. Bardeen, None; S.K.Davis, None.

POS-L54 BOARD #LB54Forced unraveling of nucleosomes assembled on heterogeneous DNA using core his-tones, NAP-1, and ACF. Gregory Gemmen, Ronald Sim, Karl Haushalter, Pu ChunKe, James Kadonaga, Douglas Smith. UCSD, La Jolla, CA, USA.Nucleosomes were assembled on λ DNA using core histones, the histone chaperone NAP-1, and ATP-dependent chromatin assembly and remodeling factor (ACF). The mechani-cal properties of these complexes were interrogated by stretching them with optical tweez-ers. Abrupt events releasing ~55 to 95 base pairs of DNA, attributable to the non-equilib-rium unraveling of individual nucleosomes, were frequently observed. This finding is com-parable with a previous observation of ~80 bp events for nucleosomes assembled by salt dial-ysis on a repeating sea urchin 5S RNA positioning element, but it differs from resultsobtained with complexes formed by exposing single λ DNA molecules to Xenopus eggextracts. Unraveling forces varied over a wider range (~5 to 65 pN) than was observed forrepeats of positioned nucleosomes. We attribute this variation to a dependence of the unrav-eling force on the DNA sequence within individual nucleosomes. The mean force increasedfrom 24 to 31 pN as NaCl was decreased from 100 to 5 mM. Spontaneous DNA re-wrap-ping events were occasionally observed during force relaxation. The observed variations inthe dynamic force needed to unravel individual nucleosomes and the occurrences of sud-den DNA re-wrapping events may have an important regulatory influence on DNA-direct-ed nuclear processes, such as the binding of transcription factors and the movement of poly-

merase complexes on chromatin. Author Disclosure Block: G. Gemmen, None; R. Sim,None; K. Haushalter, None; P. Ke, None; J. Kadonaga, None; D. Smith, None.

POS-L55 BOARD #LB55Eukaryotic genome packaging revealed by Atomic Force Microscopy II: Chicken ery-throcyte. Toshiro Kobori1, Kunio Takeyasu1, Toshio Ohtani2. 1Kyoto University, Kyoto,Japan, 2National Food Research Institute, Tsukuba, Japan.The proper function of the genome largely depends on the higher-order architectures of thechromosome. To elucidate such higher-order architectures, we have developed an ‘on-sub-strate procedure’ for subcellular fractionation of tissue-cultured human cells (Yoshimura etal., J. Electron Microsc., 52, 415-423, 2003). This procedure allows us to successivelyremove the plasma and subcellular membranes, the cytoplasm, and the nucleoplasm, and,thus, being suitable for a close look at the intra-cellular cytoskeletons and the chromatinarchitectures with a nanoscale imaging technique, atomic force microscopy (AFM). In thisreport, we applied this technique to the structural analysis of chicken erythrocyte chro-matin. Chicken erythrocyte cells on a coverslip were successively treated with buffers con-taining detergents and high-salts. After a removal of the membranes and cytoplasm with0.5% NP40, the surface of the nucleus appeared. A subsequent detergent treatment (0.5%Triton X-100) enabled a close inspection inside the nucleus. After nuclearplasm was extract-ed with a high-salt solution (250 mM (NH4)2SO4), chromatin was released from thenuclear region as a fibrous network on the substrate. The width of the fibers was 40 nm.The 40 nm fibers turned into the ‘beads-on-a-string’ structures upon the treatment with400 mM NaCl, which is known to extract the linker histone, H1, from the chicken ery-throcyte chromatin. According to our previous observations regarding yeast chromatin thathas no H1 (Kobori et al., Biophys. J., Abstract issue, 2004), the same salt treatment (400mM NaCl) induced the phase transition of chromatin structure. Such a transition was notobserved in the chicken chromatin. Therefore, H1 and linker DNA would be possibledeterminants for a physical nature of chromatin. Author Disclosure Block: T. Kobori, None;K. Takeyasu, None; T. Ohtani, None.

POS-L56 BOARD #LB56Quantitative analysis of the role of centromere chromatin organization in mitotic chro-mosome condensation. Paul S. Maddox, Arshad Desai, Karen Oegema. LICR/UCSD,La Jolla, CA, USA.Upon entry into mitosis, chromosomes condense ~20,000 fold forming discrete chromatinpackages which can be easily segregated. Microtubules of the mitotic spindle provide forceproduction for chromosome segregation by attaching to the centromere regions on chro-mosomes (via kinetochores). Therefore centromeric chromatin serves two main roles inmitosis; nucleating kinetochore assembly and acting as a “stress point” on chromosomes.To analyze the mechanistic link between kinetochore assembly and chromosome conden-sation, we developed an in vivo assay to quantify levels of chromosome condensation dur-ing the first mitotic division in C. elegans. Using this assay, we show that chromosomes incontrol embryos follow a predictable, biphasic condensation profile, with maximum con-densation occurring ~50 seconds prior to nuclear envelop breakdown (NEBD). Consistentwith a role for centromere chromatin in condensation, RNAi of CENP-A (centromere spe-cific histone H3 variant) resulted in an aberrant condensation profile. Interestingly thisdefect was specific to CENP-A, as RNAi of CENP-C (a protein required directly down-stream of CENP-A for kinetochore assembly) had no effect on chromosome condensation.The defect observed in the absence of CENP-A was unique compared to RNAi of SMC-4 (a component of the condensin complex). Therefore, our data provide a quantitative linkbetween chromosome condensation and kinetochore assembly. Author Disclosure Block:P.S. Maddox, None; A. Desai, None; K. Oegema, None.

POS-L57 BOARD #LB57One-Dimensional Transport of DNA Confined within Polymer Nano-Tubes.Ana M. Jofre, Rani Kishore, Kristian Helmerson. NIST, Gaithersburg, MD, USA.We have recently developed a technique for creating rigid cross-linked polymer nano-tubestructures, whose inner diameter are less than 50nm. Such structures can be used to con-fine long polymers in order to study one-dimensional transport properties. A long polymermolecule is said to be confined into one-dimension if the diameter of the confining tube issmaller than the molecule’s radius of gyration, in which case the polymer molecule will beextended. We are currently studying the transport properties of DNA confined within thesetubes. Lambda DNA, intercalated with YOYO-1 dye, is driven into the nano-tube bymeans of an applied electric field, and is observed through fluorescence microscopy. Detailsof our experimental method, observation of the DNA confined within the nano-tube, aswell as progress towards measurements of the diffusion time will be reported. AuthorDisclosure Block: A.M. Jofre, None; R. Kishore, None; K. Helmerson, None.

POS-L58 BOARD #LB58CHOLESTEROL - CHITOSAN INTERACTIONS AT THE AIR -WATERINTERFACE. Hilda Parra1, Miguel A. Valdés, Dr.1, María Burboa, Dr.1, JosuéJuárez1, Francisco Goycoolea, Dr.2. 1University of Sonora, Hermosillo, Mexico, 2C I AD, A. C., Hermosillo, Mexico.Chitosan is a deacetylated derivatives of chitin consisting of -1,4-N- acetylglucosamine.Chitin is widely distributed in nature as the skeletal structure of crustaceans, insects, mush-rooms, and the cell wall of fungi. Chitosan is a cationic polyelectrolyte and possesses a vari-ety of biological activities such as host-associates anticancer activity, antifungal activity andantibacterial activity. It has been suggested that chitosan can be used as a hypercholes-terolemic agent that possibly interacts with lipids droplets in the intestine.In this work we report the interaction of cholesterol and stearic acid with chitosan and otherchitosan derivatives at the air-water interface. The isotherms of both cholesterol and stearicacid are modified due to the interaction of chitosan in the aqueous phase. The area/mole-cule is modified and is depending on the lipids concentration, the molecular weight of chi-tosan and the hydrophobicity of the chitosan derivative. Infrared measurements and molec-ular simulations suggest that the electrostatic and hydrogen bond interactions are predom-inant. Monolayers and multilayers of chitosan-cholesterol and chitosan stearic acid wereobtained with the Langmuir-Blodgett method at different pressures and analyzed by atom-

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ic force microscopy. The images show that the complexes chitosan- cholesterol and chitosanstearic acid are more homogeneus and with smaller aggregates than the corresponding oneswith pure chitosan. Quantification of the adsorbed cholesterol by the different samples wereperformed with HPLC. Author Disclosure Block: H. Parra, None; M.A. Valdés, None; M.Burboa, None; J. Juárez, None; F. Goycoolea, None.

POS-L59 BOARD #LB59The Physical Properties of Goat Milk Fat Monolayers at the Air-Water Interface. JohnP. Hagen, Ellen O’Dea. Cal Poly, San Luis Obispo, CA, USA.Lipid monolayers at the air-water interface are well-characterized model system for study-ing cell membranes. While synthetic triglyceride monolayers have been studied extensively,there are few published results for monolayers composed of natural fats. We present hereour results for monolayers composed of fat extracted from goat milk. We have determinedthe isothermal compressibility, κT, and the isobaric expansion coefficient, αt, of goat milk-fat monolayers. The fat from the milk samples was extracted by the Mojonnier method.After spreading monolayers in a Langmuir trough, isotherms were taken at 25° C and 37°C to determine κT while, isochors at 5 mN/m were measured from 25° C to 39° C to deter-mine αt. The isobaric expansion coefficient was 0.0029±.0009 1/K. The isothermal expan-sion coefficient did not vary significantly with temperature; it was 0.023 ± 0.001 m/mN at25 °C and 0.024 ± 0.001 m/mN at 37 °C. These values are comparable to the liquid-expanded phase in triolein. Author Disclosure Block: J.P. Hagen, None; E. O’Dea, None.

POS-L60 BOARD #LB60Deformation of Giant Vesicles in an external shear flow field. Thomas Franke, Dr.1,Wolfgang Schnitzler2, Stephan Herminghaus, Prof.1, Thomas Pfohl, Dr.1. 1Max-Planck-Institute for Dynamics and Self-Organisation, Goettingen, Germany, 2University of Ulm,Ulm, Germany.We describe an experimental study of phospholipid vesicles in external hydrodynamicalflow fields. We present a novel counter rotating flow chamber to apply a linear shear fieldto giant vesicles. Detection of deformation is done by conventional incident-light excitationfluorescence microscopy. The dependence of the morphology on shear rate is systematical-ly studied. As a simple parameter characterizing the deformation of the vesicle the Taylordeformation parameter can be taken.In additon we present some experimental results of giant vesicle shape deformation in non-linear shear fields of different geometry. The external shear field is calculated in a finite ele-ment method computer simulation and can be related to vesicle shape deformation. AuthorDisclosure Block: T. Franke, None; W. Schnitzler, None; S. Herminghaus, None; T. Pfohl,None.

POS-L61 BOARD #LB61Thermodynamics of the Interaction of Amphiphiles with Lipid Bilayers in the Liquid-ordered and Liquid-disordered States. Maria Joao Moreno, Ph.D., Julio L. Sampaio,Magda S. C. Abreu, Winchil L. C. Vaz, Aggregation. Universidade de Coimbra, Coimbra, Portugal.We present a detailed breakdown of the elementary steps involved in the association ofamphiphilic molecules with lipid bilayer membranes in the liquid-ordered and liquid-dis-ordered states. In light of this new reaction scheme, we analyze our previously publishedresults on the kinetics of association of NBD-DMPE (Abreu et al., 2004. Biophys. J.87:353-365) and NBD-lysoMPE (Sampaio et al., 2004. Biophys. J., submitted for publi-cation) with lipid bilayers prepared from pure POPC and from binary mixtures of POPCand cholesterol (molar ratio 1:1) and sphingomyelin and cholesterol (molar ratio 6:4). Thethermodynamics of each step in the global non-catalyzed association of these amphiphileswith the bilayers mentioned (encounter complex formation, insertion, translocation, andthe respective activation processes) are then used to discuss the thermodynamics of lipidtransfer between membranes and the translocation of lipids across a membrane. The resultsare of relevance to both these processes in the biological system and to passive permeationof amphiphiles including drugs across epithelial or endothelial cells. Author Disclosure Block:M. Moreno, None; J.L. Sampaio, None; M.S.C. Abreu, None; W.L.C. Vaz, None.

POS-L62 BOARD #LB62Reconstitution of phospholipid flip-flop from the endoplasmic reticulum of yeast.Stefanie Vehring, Adrien Schroeer, Thomas Pomorski, Andreas Herrmann. HumboldtUniversity Berlin, Berlin, Germany.Transbilayer movement of phospholipids, termed flip-flop, constitutes an essential featurein processes of membrane biogenesis, stability, and dynamics.For instance, the topology of lipid synthesis and import at the cytoplasmic leaflet of endo-plasmic reticulum (ER) necessitates phospholipid flipping to the lumenal side in order toprovide for uniform distribution and membrane growth. In mammalian ER, this process israpid in either direction in the order of milliseconds to seconds for phospholipids of allheadgroups. Reconstitution has proven that mere ER lipid composition is not sufficient fortranslocation but that this depends on the presence of membrane proteins. Although theexistence of so called “flippase(s)” has been postulated, no such dedicated proteins have beenidentified yet.Our work shows that rapid, energy independent, and bidirectional flip-flop of phospho-lipids occurs in the ER of yeast S. cerevisiae. This activity could be reconstituted into pro-teoliposomes. Employing fluorescent phospholipid analogues rapid fip-flop was charac-terised via quantifying fluorescence in the outer membrane leaflet as a measure of translo-cation. We were able to show that flipping was not only protein dependent and saturablebut occuring without energy requirement with no preference for particular phospholipidheadgroups. Fractionation of crude ER protein extracts on density gradients as well as onion exchange devices resulted in reproducible enrichment of flippase activity. Furthermore,phospholipid flip-flop was sensitive to alkylation and protease treatment of reconstitutedproteins. Author Disclosure Block: S. Vehring, None; A. Schroeer, None; T. Pomorski, None;A. Herrmann, None.

POS-L63 BOARD #LB63Cholesterol in the lipid bilayer model membrane enhances the incorporation of sphin-gosylphosphorylcholine into the membrane. Katsuko Kajiya, Hiroko Kishi, HozumiKawamichi, Saori Miwa, Sei Kobayashi. Yamaguchi University School of Medicine, Ube,Japan.A Rho-kinase-mediated Ca2+ sensitization of vascular smooth muscle (VSM) contractionplays a critical role in vasospasm. We previously identified sphingosylphosphorylcholine(SPC) as the upstream mediator for the Rho-kinase-mediated Ca2+ sensitization of VSMcontraction. Although the membrane receptors for SPC were reported as the [Ca2+]i-ele-vating G-protein-coupled receptors (GPCRs), SPC-induced contractions of intact VSMwas not accompanied with any elevation of [Ca2+]i and SPC induced contraction of mem-brane-permeabilized VSM in the absence of cytosolic GTP which is required for the acti-vation of G-proteins and thus of GPCRs. These findings are compatible with the interac-tion of SPC with the other membrane components than GPCRs or the direct interactionbetween SPC and lipid membrane, which may in turn affect the function of membraneproteins. In addition, we observed the strong link between the SPC-induced contractionand the tissue and cellular cholesterol in VSM, as presented by Kishi et al. in this meeting,suggesting the roles of the cholesterol-enriched membrane microdomain, membrane lipidraft, in the SPC-induced Ca2+-sensitization of VSM contraction.Thus, we investigated the interaction of SPC with model membranes, which are preparedwith various ratio of cholesterol: phosphatidylcholine: sphingomyelin. The surface plasmonresonance measurement (BIACORE system) revealed that cholesterol in the model mem-brane enhances the incorporation of SPC into the membrane. In addition, we previouslyreported the involvement of Src family tyrosine kinases (Src-TKs), and that eicosapen-taenoic acid (EPA) inhibited the SPC-induced VSM contraction and translocation of Fyn,one of Src-TKs to the membrane raft. Therefore we also investigated the interaction of EPAwith the membrane raft. Author Disclosure Block: K. Kajiya, None; H. Kishi, None; H.Kawamichi, None; S. Miwa, None; S. Kobayashi, None.

POS-L64 BOARD #LB64Membrane poration and lysis dynamics of single cells in suspension. Dino Di Carlo,Cristian Ionescu-Zanetti, Ph.D. UC Berkeley, Berkeley, CA, USA.Viruses, bacterial toxins, natural venoms, detergents, and phospholipases induce cell lysisthrough the colloid-osmotic mechanism. This phenomenon has previously been studied byaveraging over large cell populations, or by micropipette aspiration for vesicle model sys-tems. We present direct observations of single cell lysis dynamics enabled by a microfluidicdevice capable of local application of the lytic agent. Unlike previous cell lysis studies, con-vective mixing of the lytic agent is replaced by diffusion. This allows the observation of sta-tionary cells in suspension that retain spherical symmetry. For the smaller red blood cells,the lysis event occurs 2.5 times faster than for HeLa cells, consistent with a model of con-stant flux per membrane unit area. For HeLa cells, membrane permeabilization to the cyto-plasmic dye calcein is quickly followed by volumetric cell expansion and lysis. Both the radi-al expansion and the delay between dye leak and radial expansion are weakly related to themembrane permeabilization time scale (R = 0.66 and R = 0.75 respectively). By assumingthat isotropic expansion of the membrane area leads to increased pore size during lysis, wecalculate the time dependent change in dye concentration inside the cell. A fit to the datayields values of 0.94 ± 0.6 nm for the initial pore size and N = 4-35·106 for the number ofpores, assumed to remain constant during the fast lysis event. Author Disclosure Block: D.Di Carlo, None; C. Ionescu-Zanetti, None.

POS-L65 BOARD #LB65Dynamics of Actin-driven Thin Membrane Protrusions. Erdinc Atilgan, Ph.D., DenisWirtz, Sean Sun. Johns Hopkins University, Baltimore, MD, USA.Motile cells explore their surrounding environment by sending out thin protrusions, orfilopodia, of several hundred nanometers. The growth of the protrusions is driven by actin

bundles polymerizing inside the cell mem-brane. A formalism is presented to computethe dynamical features of the membrane pro-trusions. A simple Brownian ratchet modelallows us to compute the protrusion speed asand force/velocity curves. There is a criticalnumber of filaments needed to generate pro-trusions. The protrusion speed is completelylimited by the slow fluctuations of the cellmembrane. The speed also depends on thenumber of filaments in the actin bundle andthe overall actin monomer concentration inthe cell. Without external influences, the

filopodium can extend indefinitely up to the buckling length. Author Disclosure Block: E.Atilgan, None; D. Wirtz, None; S. Sun, None.

POS-L66 BOARD #LB66Mechanism of S-Protein mediated fusion of Coronaviruses. Patricia Eifart, AndreasHerrmann, Thomas Korte. Humboldt University Berlin, Berlin, Germany.The S-glycoprotein of coronaviruses plays the key role for virus entry and cell fusion. Both,the binding to the cell surface as well as the fusion between the envelope of the virus andthe target membrane are mediated by this large trimeric glycoprotein. However, the inter-action of the S-glycoprotein with its receptor and the fusion process are not well understood.In particular, it is not known whether the interaction with the receptor is also of relevancefor triggering of the fusion activity of the S-protein. The project aims (i) to characterize theearly phase virus infection, (ii) to identify factors priming the S-protein for fusion, (iii) tocharacterize the conformational change of the S protein and respective intermediatestowards the fusion active state, and (iv) to assign intermediates of the conformationalchange to steps of virus fusion as merger of membranes and pore formation/widening. Tocharacterize the conformational change by various biochemical and biophysical methodsincluding electron microscopy, the ectodomain as well as the full length S-Protein will be

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expressed and isolated. Different model systems will be employed to study the S-Proteinmediated membrane fusion: (i) intact viruses of different corona strains e.g. transmissiblegastroentiritis virus (TGEV) and mouse hepatitis virus (MHV), (ii) cells expressing the S-glycoprotein as e.g. SARS-CoV, and (iii) reconstituted virosomes. Various (fluorescence)assays will be employed to characterize S-mediated fusion. Those systems will be also usedto identify potential inhibitors of S-mediated fusion. Author Disclosure Block: P. Eifart,None; A. Herrmann, None; T. Korte, None.

POS-L67 BOARD #LB67Fusion of Giant Unilamellar Liposomes. Erge Edgu-Fry, Rani Kishore, KristianHelmerson, Jeeseong Hwang. NIST, Gaithersburg, MD, USA.We demonstrate the fusion of giant unilamellar POPC liposomes brought together by theuse of optical tweezers. The liposomes are formed by the electroformation method. Prior tothe electroformation process DIOC dye is mixed in with the lipids. During electroforma-tion DIOC dye attaches to the membranes of the forming liposomes. A solution of sucroseis added to the electroformation chamber and the liposomes enclose this solution as theyform. The liposomes are about 10-50 micrometers in diameter. Since it is very hard to opti-cally trap giant unilamellar liposomes the sucrose solution helps increase the trapping abil-ity by changing the index of refraction of the vesicles. After the liposomes are formed theyare collected in a flask and mixed in with HEPES buffer. The sucrose solution also makesthe vesicles heavier, giant liposomes are collected from the bottom after a few minutes oflow speed centrifuge of the flask.After two liposomes are brought together by dual optical tweezers, the fusion process is ini-tiated by a UV pulse. We excite the DIOC dye on the membranes of the liposomes andimage the process fluorescence microscopy. The polarization of the excitation light is con-tinually varied during the process in order to observe the orientation of the lipids on themembranes, before, during and after fusion. Our main aim is to demonstrate whether astalk forms during the fusion process or not. Details of our optical setup, computer controland various challenges that we have overcome will be reported. Author Disclosure Block: E.Edgu-Fry, None; R. Kishore, None; K. Helmerson, None; J. Hwang, None.

POS-L68 BOARD #LB68OPA1 requires mitofusin 1 to promote mitochondrial fusion. Sara Cipolat, OlgaMartins de Brito, Barbara Dal Zilio, Luca Scorrano. VIMM, Padova, Italy.The regulated equilibrium between mitochondrial fusion and fission is essential to main-tain integrity of the organelle. Mechanisms of mitochondrial fusion are largely uncharac-terized in mammalian cells. It is unclear whether OPA1, a dynamin-related protein of theinner membrane mutated in autosomal dominant optic atrophy, participates in fusion orfission. OPA1 promoted the formation of a branched network of elongated mitochondria,requiring the integrity of both its GTPase and C-terminal coiled-coil domain. Stable reduc-tion of OPA1 levels by RNA interference resulted in small, fragmented, and scattered mito-chondria. Levels of OPA1 did not affect mitochondrial docking, but they correlated withthe extent of fusion as measured by polyethylene glycol mitochondrial fusion assays. Agenetic analysis proved that OPA1 was unable to tubulate and fuse mitochondria lackingthe outer membrane mitofusin 1 but not mitofusin 2. Our data show that OPA1 func-tionally requires mitofusin 1 to regulate mitochondrial fusion and reveal a specific func-tional difference between mitofusin 1 and 2. Author Disclosure Block: S. Cipolat, None; O.Martins de Brito, None; B. Dal Zilio, None; L. Scorrano, None.

POS-L69 BOARD #LB69Mitofusin-2, a mitochondrial GTPase mutated in Charcot-Marie-Tooth type IIA neu-ropathy, regulates endoplasmic reticulum function and endoplasmic reticulum to mito-chondria interaction. Olga Martins de Brito1, Hsuchien Chen2, David C. Chan2, LucaScorrano1. 1VIMM, Padova, Italy, 2Caltech, Pasadena, CA, USA.Mitochondrial morphology results from the net balance between fusion and fission events.Mitofusions 1 and 2 are large GTPases belonging to the family of dynamin-related-proteinsthat mediate fusion of outer mitochondrial membranes in mammalian cells. Mitochondriaparticipate in Ca2+ signalling, modulating shape and extent of cytosolic Ca2+ transients. Wereasoned that disruption of the regulated mitochondrial network could affect Ca2+ sig-nalling.Cytosolic and suprisingly mitochondrial Ca2+ transients following passive discharge of intra-cellular stores resulted bigger in Mfn2-/- mouse embryonic fibroblasts (MEFs) as comparedto their wt counterparts. We therefore turned to a biochemical, morphological and func-tional analysis of the endoplasmic reticulum (ER) of Mfn2-/- cells. Immunoblots of subcel-lular fractions showed that MFN2 was detectable also in the light membrane fraction, cor-responding to the ER and peroxisomes. Steady state ER Ca2+ concentration resulted high-er in Mfn2-/- MEFs. These functional changes were paralleled by dramatic reorganizationof the organelle, resulting in disruption of its reticular shape. Moreover, both in situ and invitro analysis revealed marked reduction of interaction between mitochondria and ER. Incomplex, our data indicate a novel role for MFN2, mutated in CMT IIA, in regulating ERfunction and ER to mitochondria interaction. Author Disclosure Block: O. Martins de Brito,None; H. Chen, None; D.C. Chan, None; L. Scorrano, None.

POS-L70 BOARD #LB70Differential coupling of Ca2+, Sr2+ and Ba2+ to SNARE-mediated membrane fusion bydistinct isoforms of synaptotagmin. Akhil Bhalla, Ph.D due 2005, Ward C. Tucker,Ph.D. University Of Wisconsin, Madison, WI, USA.Ca2+ triggers the release of neurotransmitters and hormones from neurons and neuroen-docrine cells. The sequence of events linking the influx of Ca2+ to exocytosis is still far frombeing fully understood. Members of the synaptotagmin (syt) gene family have been pro-posed to function as the Ca2+ -sensors that regulate release however, their specific tissue dis-tribution/localization and precise function remains unclear. Previously it was shown that sytI can stimulate Ca2+ dependent membrane fusion in vitro.Syt•membrane and syt•SNAREs interactions are thought to be important for neurotrans-mitter release. While syt I•SNARE interactions are well characterized, the ability of othersyt isoforms to bind t-SNAREs remains an open issue. Classical electrophysiological stud-ies indicated that Sr2+ and Ba2+, in addition to Ca2+, are able to trigger neurotransmitter

release from different cell preparations however, the ability of different syt isoforms to senseSr2+ and Ba2+ is somewhat of a controversy. We have addressed these questions by using areduced in vitro assay for SNARE mediated membrane fusion. We asked if different syt iso-forms can trigger membrane fusion while also determining the metal sensitivities for thesereactions. I, VII and IX were found to trigger membrane fusion in a Ca2+ dependent man-ner with a 300-fold difference in [Ca2+]1/2. In addition, Ba2+ and Sr2+ triggered membranefusion in a isoform specific manner reflecting differences in affinities of these syts towardsdivalent cations. We found syts that trigger membrane fusion bind t-SNAREs that areembedded into vesicle bilayers. To further elucidate the function of syt•SNARE interac-tions, we showed that mutations that disrupt syt•SNARE interactions have a negative effecton the fusion assay. Together these data provide further evidence that syt•SNARE interac-tions are important steps that couple Ca2+ influx to neurotransmitter release. AuthorDisclosure Block: A. Bhalla, None; W.C. Tucker, None.

POS-L71 BOARD #LB71EFFECTS OF PRESSURE ON THE STRUCTURE OF TERNARY LIPID BILAY-ER SYSTEMS- A FLUORESCENCE MICROSCOPY STUDY - Chiara Nicolini1,Anna Celli2, Enrico Gratton2, Roland Winter1. 1Physical Chemistry I, University of Dortmund, Germany, 2Laboratory for FluorescenceDynamics, University of Illinois, IL, USA.Lipid systems, which provide valuable model system for biological membranes, display avariety of phases on the nanometer and micrometer scale. Substantial evidence has accu-mulated now that points to the presence of distinct lipid regions termed ”rafts” in cell mem-branes. They are rich in sphingomyelin and cholesterol and are thought to be important forcellular functions. We studied the structure and lateral organization of raft model systems,such as 1:1:1 DOPC/Sphingomyelin(or DPPC)/cholesterol using two-photon excitationfluorescence microscopy. This technique is useful for direct visualization of the microscop-ic scenario of lipid phase equilibria including information on the lipid phase state of giantunilamellar vesicles (GUVs) in the micrometer range. Hydrostatic pressure has been usedhere as a physical parameter for studying the stability and energetics of lipid phases, but alsobecause high pressure is an important feature of certain natural membrane environments.The pressure-dependent experiments were performed using a sample cell made from a thinfused silica capillary. The use of Laurdan as fluorescence label allowed us to follow the lipidphase state by calculating the GP values of the vesicles and extracting their average value.During the compression cycle, the GP value increases, indicating an increasingly tighterpacking of the lipids upon pressurization up to ~500-1000 bar, where pressure-inducedphase transitions occur. The transitions are accompanied by characteristic changes in vesi-cle size. The presence of ~33 mol% cholesterol leads to a drastic stabilization of the mem-brane system and causes less volume change (~11%) upon pressurization. No changes inthe vesicle shape were observed as we did in case of the system POPC/DLPC. The changesin GP values are compared to our pressure dependent FT-IR data on the lipid conforma-tions in MLVs. *E-mail: roland.winter@uni-dortmund.de. Author Disclosure Block: C.Nicolini, None; A. Celli, None; E. Gratton, None; R. Winter, None.

POS-L72 BOARD #LB72Effect of Membrane Characteristics on Phase Separation and Domain Formation inCholesterol-Lipid Mixtures. Veena Pata, Ph.D.1, Nily Dan, Ph.D.2. 1NIH, Bethesda,MD, USA, 2Drexel University, Philadelphia, PA, USA.We examine, using an analytical mean-field model, the distribution of cholesterol in a lipidbilayer. The model accounts for the perturbation of lipid packing induced by the embed-ded cholesterol, in a manner similar to that of trans-membrane proteins. We find that themembrane-induced interactions between embedded cholesterol molecules vary as a func-tion of the cholesterol content. Thus, the effective lipid-cholesterol interaction is concen-tration-dependent. Moreover, it transitions from repulsive to attractive to repulsive as thecholesterol content increases. As the concentration of cholesterol in the bilayer exceeds a crit-ical value, phase separation occurs. The coexistence between cholesterol-rich and choles-terol-poor domains is universal for any bilayer parameters, although the composition of thecholesterol-rich phase varies as a function of the lipid properties. Although we do notassume specific cholesterol-lipid interactions or the formation of a lipid-cholesterol cluster,we find that the composition of the cholesterol-rich domains is constant, independent ofthe cholesterol content in the bilayer. Author Disclosure Block: V. Pata, None; N. Dan, None.

POS-L73 BOARD #LB73Patterning complexity into supported planar bilayers. Erin D. Sheets, PhD, KanikaVats. Pennsylvania State University, University Park, PA, USA.We are probing the relationship between membrane structure and biological function in liv-ing cells and on biomimetic model membranes using laser-based microscopy. To accom-plish this goal, we are using a combination of microlithography and quantitative fluores-cence microscopy to systematically probe the molecular interactions that lead to lipiddomain formation as well as domain dynamics. Complexity is built into supported bilay-ers by pattering domains via a polymer lift-off approach. As we develop our approach, weare initially using two lipids: dilauroylphosphatidylcholine (DLPC), which has been dopedwith 2-BODIPY FL C5-1-hexadecanoylphosphatidylcholine, and dipalmitoylphos-phatidylcholine (DPPC), which has been doped with DiI C18(3). This lipid composition,which is immiscible at room temperature, allows us to easily characterize our constructedpattern. We first create arrays of 5 um or 10 um squares of a particular lipid bilayer (e.g.,DLPC), which is supported on a glass substrate. Complexity is then introduced by subse-quent incubation with the other component (e.g., DPPC). We find that the complex bilay-ers retain their patterns, and that multilayers do not form, as assessed by quantitative fluo-rescence microscopy. Atomic force microscopy and fluorescence correlation spectroscopy(FCS) are being used to further characterize these patterned bilayers. We will also use singleparticle tracking and FCS, as well as imaging, to probe the molecular dynamics of lipids inthe domains and at the interfaces between the domains with high spatial and temporal res-olution. Ultimately, we will follow the dynamics of domains and individual lipids withinthis milieux as a function of cholesterol content because we want to understand how these

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physical interactions (cholesterol-rich rafts) control biological function. Our approach mayalso allow us to construct much improved biosensors. Author Disclosure Block: E.D. Sheets,None; K. Vats, None.

POS-L74 BOARD #LB74Blebs as an Indicator of Malignancy Transformation. Michelle Khine, PhD, CristianIonescu-Zanetti, PhD, Luke P. Lee, PhD. UC Berkeley, Berkeley, CA, USA. Keywords:cell mechanics, blebs, malignancy.A cell’s mechanical behavior can also offer much insight into its overall health. For exam-ple, cancerous cells differ from normal cells in that they display reduced stiffness, allowingthem to travel through capillaries and spread throughout the body in the metastatic stage.Therefore, the mechanical properties of cells can be used as a valuable diagnostic tool tostudy cancer. During the transformation process to malignancy, the cytoskeleton progress-es from being rigid and organized to being softer and more irregular. This is accompaniedby a marked decrease in the amount of cytoskeletal actin filaments. Because the shear mod-ulus of actin filament solution is non-linearly proportional to its actin concentration, evena slight decrease in actin would therefore translate into a marked reduction in elasticity.Studies are now using a cell’s rigidity as a biological marker for cell function. Dynamic forcespectroscopy can also be used to shed light on the properties of certain cells. The needremains for a fast and efficient way to rapidly screen large numbers of cells.We are working on correlating membrane perturbations with cell types. Empirical obser-vations have indicated that cells that are more transformed tend to exhibit more hernia-likelocal dissociations of the cell membrane from the cytoskeleton, known as blebs, whenstressed as compared to non-malignant cells. To quantify the mechanical behavior of thecell, each cell is drawn into a channel with a diameter smaller than that of the cell radius.The resulting deformation (elongation) is measured as a function of pressure and time,offering a measure of both the elastic (Young’s modulus) and viscous properties of the cellbeing tested. Bleb dynamics (size, number, rate of formation) as a function of deformationis quantified and then compared across cell types. Author Disclosure Block: M. Khine,None; C. Ionescu-Zanetti, None; L.P. Lee, None.

POS-L75 BOARD #LB75Membrane Perturbation Effects of Peptides Derived from the N-termini ofUnprocessed Prion Proteins. Mazin Magzoub1, Kamila Oglecka1, Aladdin Pramanik,PhD2, L. E. G. Eriksson1, Astrid Gräslund1. 1Biochemistry and Biophysics, Stockholm,Sweden, 2Medical Biochemistry and Biophysics, Karolinska Institute, Sweden.Peptides derived from the unprocessed N-termini of mouse and bovine prion proteins(mPrPp and bPrPp, respectively), comprising hydrophobic signal sequences followed bycharged domains (KKRPKP), function as cell-penetrating peptides (CPPs) with live cells,concomitantly causing toxicity. Using fluorescence correlation spectroscopy (FCS), we stud-ied the membrane interactions of the peptides with large phospholipid vesicles (LUVs),either rhodamine-entrapping (REVs) or rhodamine-labeled (RLVs), and compared theeffects with those of the CPP penetratin (pAntp) and the lytic peptide melittin. LUV mem-brane interactions of the peptides were further characterized using fluorescence polarizationof a membrane probe (DPH), calcein leakage, and circular dichroism. The FCS resultsshow that the prion peptides, like melittin, cause rhodamine release from REVs, withoutaffecting the overall integrity of RLVs. Increased membrane ordering and calcein leakagecaused by the prion peptides followed the order of potency for rhodamine release by FCS:melittin > mPrPp > bPrPp> pAntp. Upon addition of pAntp to fully charged RLVs, FCSdata revealed a time dependent irreversible aggregation of the vesicles, related to inducedmembrane perturbation. The membrane perturbation effects of the N-terminal prion pep-tides suggest that they form transient pores, similar to melittin, causing toxicity, in parallelwith their cellular trafficking. Author Disclosure Block: M. Magzoub, None; K. Oglecka,None; A. Pramanik, None; L. Eriksson, None; A. Gräslund, None.

POS-L76 BOARD #LB76AβP(1-40)- channels are sensitive to divalent cations in reconstituted oxidized choles-terol planar lipid membranes. Silvia Micelli, professor, Laura La Stella, PHD, DanielaMeleleo, PHD, Enrico Gallucci, Professor. University, Bari, Italy.It has been previously been demonstrated that AβP easily incorporates easily into oxidizedcholesterol (OxCh) (and sterols in general) membranes (and sterols in general) to formanion-selective channels (Micelli et al., BJ 2004,86:2231-7). Oxidized Cholesterol (OxCh)membranes, with some allowances, can be used as a model to simulate in vivo conditionsof aged membranes. In this study, we report the effect of divalent cations, namely Ca2+,Ba2+, Mg2+, Zn2+, on the conductance, life time and occurrence of AβP (1-4040)- chan-nels reconstituted in OxCh PLMs. The results indicate that the addition of low divalentcations concentrations (0.25 mM) on the same side of as AβP, causes an immediately blocksof channel activity. A Partial channel activity can be restored upon application of potentialas high as 140mV. These results are in accordance with those reported by Arispe et al. andKawahara et al. (PNAS 1996,93:1710-15; BJ 1997,73:67-75) on Zn2+ interaction withAβP(1-40)- channels reconstituted in phospholipids membranes and excised membranepatches from hypothalamic neurons. Author Disclosure Block: S. Micelli, None; L. LaStella, None; D. Meleleo, None; E. Gallucci, None.

POS-L77 BOARD #LB77Allosterism in Membrane Binding: a Common Motif of the Annexins? Paulo F. F.Almeida1, Katie A. Rasch2, Catherine M. Wieser2, Adekunle I. Elegbede2, HitoshiSohma3, Anne Hinderliter2. 1University of North Carolina, Wilmington, Wilmington,NC, USA, 2North Dakota State University, Fargo, ND, USA, 3Sapporo Medical School,Sapporo, Japan.Annexins are described as a family of proteins that is Ca2+-dependent for phospholipidbinding. Yet, annexins have a wide variety of binding behaviors and conformational states,some of which are lipid-dependent and Ca2+-independent. We present a model that cap-tures the cation and phospholipid binding behavior of the highly conserved core of theannexins. Experimental data for annexins A4 and A5, which have short N-termini, wasglobally modeled to gain an understanding of how the lipid-binding ability of the conserved

protein core is modulated. Analysis of the binding behavior was achieved through use ofthe lanthanide Tb3+ as a Ca2+ analogue. Binding isotherms were determined experimental-ly from the quenching of the intrinsic fluorescence of annexins A4 and A5 by Tb3+ in thepresence or absence of membranes. In the presence of lipid, the affinity of annexin for cationincreases and the binding isotherms change from hyperbolic to weakly sigmoidal. Thisbehavior was modeled by isotherms derived from microscopic binding partition functions.The change from hyperbolic to sigmoidal binding occurs because of an allosteric transitionfrom the annexin solution state to its membrane-associated state. Binding of lipid renderscation binding by annexins cooperative. The two annexin states denote two affinities of theprotein for cation, one in the absence and another in the presence of membrane. In theframework of this model, we discuss membrane binding as well as the influence of the N-terminus in modifying the annexin cation-binding affinity by changing the probability ofthe protein to undergo the postulated two-state transition. Author Disclosure Block: P.F.F.Almeida, None; K.A. Rasch, None; C.M. Wieser, None; A.I. Elegbede, None; H. Sohma,None; A. Hinderliter, None.

POS-L78 BOARD #LB78Myo1c Tail Binds PIP2 in Lipid Vesicles. David E. Hokanson, E. M. Ostap, PhD.University of Pennsylvania School of Medicine, Philadelphia, PA, USA.Myo1c is a single-headed unconventional myosin-I that associates with the plasma mem-brane. Myo1c contains a single catalytic motor domain, three IQ domains, and a basic taildomain. Previous experiments have shown that myo1c binds acidic phospholipids througha basic tail domain via electrostatic interactions. This association with the membrane isthought to be important for both localization and function. We measured binding of anexpressed protein construct consisting of the tail domain plus IQs to sucrose-loaded 100nm unilamellar vesicles (LUVs) composed of PC and various concentrations of PIP2 andPS via sedimentation assays. The tail domain binds tightly to LUVs containing > 60% PS(KB < 5 μM), but very weakly to LUVs containing < 20% PS (KB > 500 μM). Interestingly,only 2% PIP2 in PC LUVs is required for tight binding (KB < 19μM), whereas LUVs con-taining PS with a similar charge as the PIP2 LUVs (6% PS) show no detectable binding. Ithas been proposed that a single myosin I tail binds multiple PS molecules. Thus our datasuggest that it is energetically unfavorable for the myo1c tail to cluster PS when it is pres-ent in low concentration in LUVs. However, myo1c binds tightly to LUVs containing lowconcentrations of PIP2, which has a negative trivalent charge. To investigate this further,competition experiments were done with both PLCδ PH domain, which binds specifical-ly to PIP2, and negatively trivalent IP3. PLCδ PH domain competed off the tail for PIP2,while IP3 was able to compete with PIP2 and PS for the tail. We also investigated myo1ctail association to PS and PIP2 LUVs in the presence of CaM and calcium. AuthorDisclosure Block: D.E. Hokanson, None; E.M. Ostap, None.

POS-L79 BOARD #LB79Probing the behavior of small unilamellar lecithin-cholesterol model biliary vesicles toaid in understanding the gallstone pathway. Manasa V. Gudheti, Ph.D., Steven P.Wrenn, Ph.D.. Drexel University, Philadelphia, PA, USA.We utilize fluorescence spectroscopy, absorbance, dynamic light scattering and cryo-TEMto study the behavior of small unilamellar lecithin-cholesterol vesicles (~50-100 nm) in anattempt to understand gallstone pathogenesis. The focus of this research effort is to probethe combined interaction of phospholipase C (PLC), a pro-nucleating enzyme, andapolipoprotein A-I (apo A-I), an anti-nucleating protein, with vesicles. Preliminary resultsindicated that this combined interaction initiates the formation of a newly discoveredmicrostructure in bile, namely the apo A-I/lecithin/cholesterol complex (~15 nm). Withthe aid of Förster resonance energy transfer from dehydroergosterol (DHE), a fluorescentanalog of cholesterol, to dansylated lecithin (DL), a fluorescent analog of lecithin, we offerfurther proof for the existence of the complexes and characterize the dose-dependent effectof cholesterol mole fraction in the vesicles and apo A-I concentration on complex forma-tion.Results indicate that apo A-I inhibits cholesterol nucleation by two mechanisms. Initially,protection is achieved by apo A-I shielding of hydrophobic diacylglycerol (DAG) moietiesand hence prevention of vesicle aggregation. Protection by shielding is however temporary,however, and lost when the DAG/apo A-I exceeds a critical value. Subsequently, apo A-Iforms the aforementioned complexes. The critical value at which this microstructural tran-sition occurs depends on the relative concentrations of apo A-I and PLC and the choles-terol mole fraction of the vesicles. By coupling the experimental results with a modifiedMichaelis-Menten enzyme kinetic model, we are able to characterize vesicle behavior as afunction of DAG/bound apo A-I ratio. Author Disclosure Block: M.V. Gudheti, None; S.P.Wrenn, None.

POS-L80 BOARD #LB80A preference for PIP2 drives PKC alpha C2 domain translocation to PIP2-enrichedlipid rafts in the plasma membrane. John H. Evans1, Christina C. Leslie2, Joseph J.Falke1. 1University of Colorado, Boulder, CO, USA, 2National Jewish Medical andResearch Center, Denver, CO, USA.Targeting of the ubiquitous signaling molecule protein kinase C alpha (PKCα) to the innerleaflet of the plasma membrane in response to intracellular Ca2+ signals is a function of itscalcium-dependent lipid-binding or C2 domain. The goal of the work presented here is toidentify the phospholipid preference and the precise intracellular targeting of the isolatedPKCα C2 domain and to correlate both phospholipid preference and intracellular target-ing with specific residues and structures.Protein/vesicle FRET analysis demonstrated a Ca2+-dependent preference of wild-typePKCα C2 protein for phosphatidylinositol-4,5-bisphosphate (PIP2)-containing phospho-lipid vesicles. Time-lapse epifluorescence microscopy of a fluorescent protein/PKCαC2fusion revealed targeting to PIP2-rich lipid rafts in the plasma membrane in response tointracellular Ca2+ mobilization. In contrast, mutation of lysine residues in a basic regionformed by the PKCαC2 β3 and 4 strands abrogated the preference for PIP2-containingvesicles as well as the exclusive intracellular targeting to PIP2-rich lipid rafts.A cPLA2C2/PKCαC2 domain hybrid, in which the calcium binding loops (CBLs) from

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PKCαC2 replaced those of cPLA2C2 (which targets Golgi/ER), failed to demonstrate apreference for PIP2-containing phospholipid vesicles and failed to exhibit exclusive target-ing of lipid rafts. However, a second cPLA2C2/PKCαC2 hybrid, containing the PKCαC2CBLs and β3 and 4 strands, displayed a preference for PIP2-containing phospholipid vesi-cles and targeted lipid rafts in the plasma membrane in a manner similar to the wild-typePKCα C2 domain.These results suggest that the basic region formed by the PKCαC2 β3 and 4 strands specif-ically recognizes PIP2 and is critical for proper targeting of PKCα to PIP2-rich lipid rafts inthe plasma membrane. Author Disclosure Block: J.H. Evans, None; C.C. Leslie, None; J.J.Falke, None.

POS-L81 BOARD #LB81Low Mobility of PIP2 in the Plasma Membrane. Hana Cho1, Suk-Ho Lee2, Won-Kyung Ho2. 1Kangwon National University College of Medicine, Chunchon, Republicof Korea, 2Seoul National University College of Medicine, Seoul, Republic of Korea.We have shown in the previous study that cardiac GIRK channels are inhibited by Gq pro-tein coupled receptors via phosphatidylinositol 4,5-bisphosphate (PIP2) depletion in areceptor-specific manner. One hypothesis to explain signaling specificities is the localizationof signaling molecules due to low mobility. To address whether this idea can be applied toPIP2 signaling, we analyzed the lateral diffusion of bodipy-FL PIP2 in the plasma membraneby the fluorescence recovery after photobleaching (FRAP) technique. When PIP2 wasloaded in cardiomyocytes or HEK 293 cells, the fluorescence labels are equally found in thecytosol and membrane. The FRAP rate for PIP2 was as fast as that for DiI, a freely diffusiblemembrane label whose diffusion coefficient (D) was ~10-8 cm2/s. To make the conditionmore reliable for measuring lateral diffusion along the plasma membrane, we tried to washout fluorescence labels in the cytosol by making whole cell using a patch electrode. Duringseveral minutes after patch break-in, fluorescence signals from cytosol decreased gradually,while signals from surface membrane were unaffected. In this configuration, the FRAP ratefor PIP2 was significantly delayed, and D for PIP2 was calculated to be ~10-12 cm2/s. Weconfirmed that patch break-in did not affect diffusion rate of DiI. We supposed that thereis a special mechanism for slowing mobility of PIP2 in cardiomyocytes and HEK 293 cells,and tested whether the cytoskeleton is responsible for localizing PIP2. When cytochalasinD, actin disrupting agent, was pretreated, the FRAP rate for PIP2 measured after patchbreak-in was increased significantly, and D was calculated to be ~10-8 cm2/s. Taken togeth-er, it is suggested that lateral diffusion of PIP2 in cardiomyocytes and HEK 293 cells is slowand cytoskeleton is responsible for the low mobility of PIP2. Author Disclosure Block: H.Cho, None; S. Lee, None; W. Ho, None.

POS-L82 BOARD #LB82Crystal Structures of the GluR5 and GluR6 Ligand Binding Cores: MolecularMechanisms Underlying Kainate Receptor Selectivity. Mark Mayer, Ph.D. NIH,Bethesda, MD, USA.Ionotropic glutamate receptors are ligand-gated ion channels that mediate fast synaptictransmission in the CNS. In humans iGluRs are encoded by 18 genes which coassemble toform AMPA, kainate and NMDA subtype glutamate receptors. This classification was pro-posed 25 years ago following the discovery of subtype selective agonists but little is knownabout the underlying molecular mechanisms. Crystal structures of the GluR5 and GluR6kainate receptor ligand binding cores in complexes with glutamate, 2S,4R-4-methylgluta-mate, kainate, and quisqualate have now been solved. The structures reveal that the ligandbinding cavities are 40% (GluR5) and 16% (GluR6) larger than for the AMPA receptorGluR2 and contain trapped water molecules absent from the GluR2 structures reportedpreviously by Armstrong & Gouaux. The binding of AMPA and GluR5 selective agoniststo GluR6 is prevented by steric occlusion which also interferes with the binding of 2S,4R-4-methylglutamate to AMPA receptors, a ligand which binds to GluR5 and GluR6 with a1000-fold higher affinity than to GluR2. Strikingly, the extent of domain closure producedby the GluR6 partial agonist kainate is only 3° less than for glutamate and 11° greater thanfor the GluR2 kainate complex. Kainate is a much more efficacious agonist at GluR6 indi-cating that the mode of action of partial agonists at kainate receptors is regulated by domainclosure, as first described for AMPA receptors, and distinct from the behaviour of NMDAreceptors. The high affinity of kainate for GluR5 and GluR6 likely results from this greaterdomain closure which permits formation of extensive interdomain contacts betweendomains 1 and which are absent from AMPA receptors. This crystallographic analysis addsto our rapidly growing knowledge of the molecular mechanisms underlying the function ofligand gated ion channels. Author Disclosure Block: M. Mayer, None.

POS-L83 BOARD #LB83Effects of cAMP stimulation on efficiency of galvanotaxis in Dictyostelium discoideum.Masayuki J. Sato, Masahiro Ueda, Hiroaki Takagi, Toshio Yanagida. Osaka University,Suita-shi, Japan.To investigate the mechanism of directional cell migration to extracellular stimulus, it isimportant to set up experimental system in which the controlled and uniform gradients ofstimulus can be generated precisely in a highly reproducible manner. Directed current elec-trical fields (dcEF) are useful to generate such gradients of stimulus. A major cellular effectof dcEFs is galvanotaxis, which is directional movement of cells towards the cathode or theanode. Galvanotaxis provide a convenient model system for investigating basic aspects ofdirectional cell motility. We have developed the experimental system which can generateelectrical fields at a high speed and low noise and analyze the cell migration.Dictyostelium discoideum is a model organism in which the signaling pathway drivingchemotaxis has been well characterized. It has already known that Dictyostelium cell canexhibit galvanotaxis toward cathodal face. (Zhao M.et.al.,2002) We have applied our exper-imental system to Dictyostelium galvanotaxis. The efficiency of galvanotaxis was voltagedependent, while cell migration speed remained almost constant. We found that the effi-ciency of galvanotaxis was proportional to the square of the EF strength. Next, the effect ofcAMP on galvanotaxis was investigated. cAMP is an attractant of Dictyostelium cells. It hasknown that cAMP binds to the receptor and activate chemotactic signaling pathway. Thegalvanotactic efficiency in the presence of cAMP was about 1.6 times larger than that in the

absence of cAMP, suggesting that the activation of chemotactic signaling pathway enhancethe sensing efficiency of electrical field direction. Author Disclosure Block: M.J. Sato, None;M. Ueda, None; H. Takagi, None; T. Yanagida, None.

POS-L84 BOARD #LB84Frequency-encoded Nuclear Ca2+ Signals in Airway Epithelial Ciliated Cells. PedroVerdugo, MD, Ivan Quesada, PhD. University of Washington, Friday Harbor, WA,USA.The phenomenology of nuclear [Ca2+] dynamics has experienced important progressrevealing the broad range of cellular processes that it regulates. Although several agonists canmobilize Ca2+ from the nuclear envelope storage (NE) to the intranuclear compartment(INC), the mechanisms of [Ca2+] fluctuations in the nucleus still remain uncertain. Herewe report the first demonstration that the NE/INC complex can function as an inositol-1,4,5-trisphosphate (InsP3)-controlled Ca2+ oscillator. Experiments conducted in culturedairway epithelial ciliated cells or in their isolated nuclei indicate that in intact cells ATP cantrigger ~0.2 Hz periodic oscillations of [Ca2+] in the NE ([Ca2+]NE) and correspondingpulses of Ca2+ release to the INC. Identical results were obtained in InsP3-stimulated iso-lated nuclei. [Ca2+] oscillations in the INC/NE complex result from the interplay betweenthe Ca2+/K+ ion-exchange properties of the NE polyanionic matrix and two Ca2+-sensitiveion channels?an InsP3-receptor-Ca2+ channel, and an apamin-sensitive K+ channel. A sim-ilar mechanism controls [Ca2+] oscillations and release to the cytosol in/from the endoplas-mic reticulum and secretory granules (Nguyen et al. (1998) Nature 395, 908-912; Quesadaet al. (2001) Biophys. J. 80, 2133-2139; Quesada et al. (2004) Biophys. J. 85, 963-970).Our studies demonstrate that frequency-encoded intracellular Ca2+-signaling is controlledby a highly conserved mechanism that allows the nucleus and other organelles to functionas InsP3-controlled local Ca2+ oscillators, independent from cytosolic Ca2+ signaling.Author Disclosure Block: P. Verdugo, None; I. Quesada, None.

POS-L85 BOARD #LB85Structural insight into the molecular mechanisms of SRP targeting and release. PamelaJ. Focia, Joseph Gawronski-Salerno, John S. Coon, V, Douglas M. Freymann.Northwestern University, Chicago, IL, USA.Two structurally homologous Signal Recognition Particle GTPases Ffh (SRP54) & FtsY(SR?) interact directly during co-translational targeting to the membrane. Assembly of thetargeting complex is primed by GTP binding to both proteins, and, subsequent to releaseof the translating ribosome to the translocon, GTP hydrolysis drives their disengagement.The structure of the GMPPCP stabilized complex of the ‘NG’ GTPase domains ofprokaryotic SRP GTPases Ffh and FtsY [1] reveals an extensive and symmetric interfacethat buries both active sites from solvent and couples to an interdomain shift that may actallosterically to signal assembly to accessory domains of the targeting complex. The presenceof several highly conserved surface-exposed regions suggests how specific interactions withother elements of the targeting complex may regulate stimulation of GTP hydrolysis to dis-engage this molecular ‘latch’. Recent structural data provides additional insight into thesemechanisms. [1] Focia et al (2004) Science 303 373. Author Disclosure Block: P.J. Focia,None; J. Gawronski-Salerno, None; J.S. Coon, None; D.M. Freymann, None.

POS-L86 BOARD #LB86Ca2+ stores and CaM Kinase II mediate activity-induced synaptic peptidergic vesiclemobilization. Dinara Shakiryanova, Edwin S. Levitan. University of Pittsburgh,Pittsburgh, PA, USA.in vivo imaging of a GFP-tagged neuropeptide (atrial natriuretic factor) in Drosophila lar-val neuromuscular junctions shows that the motion of peptidergic dense-core vesicles(DCVs) in synaptic boutons increases in response to electrical activity. This mobilizationpersists for minutes and is accompanied by posttetanic potentiation of neuropeptide release.Here we investigate the role of intracellular Ca2+ stores in activity-dependent DCV mobi-lization.Depletion of Ca2+ stores by inhibiting SERCA Ca2+-ATPase with thapsigargin or cyclopi-azonic acid prevented activity-induced mobilization of DCVs. Blockade of inositol 1,4,5-trisphosphate (IP3) receptors with xestospongin C or ryanodine receptors with ryanodinealso abolished activity-induced mobilization of neuropeptidergic vesicles. Caffeine, a stim-ulator of Ca2+-induced Ca2+ release via ryanodine receptors, increased motion of synapticDCVs and evoked neuropeptide release in the absence of extracellular Ca2+, and theseeffects were prevented by ryanodine. Ca2+ imaging of boutons expressing the cameleonCa2+ indicator revealed that activity-induced Ca2+ elevation is not sustained. Therefore, per-sistent DCV mobilization is only triggered by Ca2+ release. DCV mobilization was alsoreduced after inhibition of Ca2+/calmodulin-dependent kinase II (CamK II) by KN-62 orKN-93, but unaffected by the inactive analog KN-92. These data indicate that Ca2+ releasefrom endoplasmic reticulum via IP3 and ryanodine receptor channels triggers DCV mobi-lization. Furthermore, CamK II is essential for increasing DCV mobility by linking tran-sient increases of intracellular Ca2+ to persistent mobilization of synaptic DCVs. Supportedby NIH grant NS32385. Author Disclosure Block: D. Shakiryanova, None; E.S. Levitan,None.

POS-L87 BOARD #LB87Dialkylcarbocyanines for Multiple Labeling of Red Blood Cell Populations for In VivoSurvival Studies. Jonathan K. Armstrong, PhD CSci CChem MRSC, Rosalinda B.Wenby, BS, Herbert J. Meiselman, ScD, Timothy C. Fisher, MB ChB. Keck School ofMedicine, University of Southern California, Los Angeles, CA, USA.Background: Measurement of the circulating lifetime of red blood cells (RBCs) requireslabeling with a radioisotope, fluorescent probe or affinity tag. Limitations of existing tech-niques include: (i) progressive loss of the probe or transfer to other cells; (ii) variation in themeasured clearance rate with different labels; (iii) immune responses to the labeled cells.Also, only one population is observed at a time. To address these limitations, we developedand evaluated a technique to label multiple populations of RBCs using dialkylcarbocyaninefluorescent dyes (Vybrant™ DiO, DiI and DiD). These lipophilic dyes are bright, stableand are easily observed by flow cytometry. Methods: in vitro studies: Human RBCs wereincubated at 37°C at a 10% hematocrit in HEPES buffer with freshly prepared 1, 4 or 8

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μM solution of an individual Vybrant dye or a mixture of two of the three dyes, giving 36combinations in total. In vivo studies: Autologous rabbit RBCs were stained in the samemanner and reinfused. Blood samples were taken over 5 weeks and analyzed by flow cytom-etry. Results: In vitro, up to fifteen discrete populations could be distinguished by flowcytometry. In vivo evaluation of 8 RBC populations showed normal survival. A minorreduction (10-30%) in fluorescence intensity was noted over the study period which didnot compromise the identification of each population. Repeated exposure to Vybrant-labeled RBCs (up to 15 exposures over 3 years) showed no evidence of significant immuno-genicity. Conclusion: The use of dialkylcarbocyanines is ideal for studies which require mul-tiple RBC populations to be followed within the same experiment, and may also have appli-cation for different cell types or particles, e.g., platelets or liposomes. Author DisclosureBlock: J.K. Armstrong, None; R.B. Wenby, None; H.J. Meiselman, None; T.C. Fisher, None.

POS-L88 BOARD #LB88PKA Increases Plasma Membrane Expression of KCNQ1 in Airway Epithelial Cells viaan AKAP-Dependent Mechanism. Steve M. Hoynowski, MS, Kathy E. Mitchell, PhD.University of Kansas, Lawrence, KS, USA.The KCNQ1 potassium channel is expressed abundantly in epithelia, along with accesso-ry subunits of the KCNE family. KCNQ1 is expressed at the basolateral surface in airwayepithelial cells where it associates with KCNE3. In the heart, β-adrenergic stimulationincreases the activity of the KCNQ1/E1 channel. This is mediated via PKA activation andrequires the A-kinase anchoring protein (AKAP), yotiao. PKA activation also increasesKCNQ1/E3 currents in epithelial cells. The mechanism of this PKA enhancement of chan-nel activity is not known. We hypothesize that PKA modulation of KCNQ1 activity in theairway epithelial cells, as for heart , is largely mediated via AKAPs, which play a role in traf-ficking of KCNQ1 to the plasma membrane. Here we show that activation of PKA bytreatment with β-adrenergic agonists or forskolin results in increased expression of KCNQ1at the plasma membrane of Calu-3 cells, a human airway epithelial cell line. The increasedtrafficking to the plasma membrane can be blocked by pretreatment with St-Ht31, a pep-tide that disrupts the association of the PKA RII subunit with AKAPs or by PKA inhibitors.Using whole cell patch-clamp analysis we show that Calu-3 cells have a forskolin-activatedcurrent that can be blocked by Xe-991, a specific KCNQ1 inhibitor. This current is absentwhen Calu-3 cells are dialyzed with the St-Ht31 in the patch pipette. We also show that thePKA-RII subunit and KCNQ1 can be co-immunoprecipitated from rat lung and Calu-3cells. These results suggest that a yet to be identified AKAP associates with KCNQ1 in air-way epithelial cells. We conclude that PKA activation increases KCNQ1 channel activity inairway epithelial cells primarily via an AKAP-dependent increase in plasma membraneexpression of KCNQ1. Supported by P20 RR017686. Author Disclosure Block: S.M.Hoynowski, None; K.E. Mitchell, OB/Gyn CellSecure Receipt of royalties.

POS-L89 BOARD #LB89MITOCHONDRIAL DYNAMICS: INNER MEMBRANE PROTEINS ARESWIPED DURING NORMAL FUSION OF MITOCHONDRIA. Karin B. Busch,PhD, Marina Jendrach, PhD, Juergen Bereiter-Hahn, PhD. Institute of Kinematic Cell Research, Frankfurt, Germany.Mitochondria possess a dynamic morphology, their appearance reach from long tubular toshort round ones. This is due to mitochondrial fission and fusion, which are regular eventsin many cultured mammalian and even plant cells. A possible rationale for these processescould be that fusion acts as a rescue mechanism for damaged mitochondria, e.g. comple-mentation of mitochondrial defects had been achieved by exchange of mtDNA. We areinterested whether also mitochondrial proteins are exchanged. To address this, a proteinlocated in the inner mitochondrial membrane was tagged with a green and red fluorescentmarker, respectively. Differently labelled cells were fused and mitochondrial fusion wasmonitored by confocal laser scanning microscopy. Here we can show for the first time thatproteins of the inner mitochondrial membrane can be exchanged between different mito-chondria. By now, three proteins (mfn1, mfn2 and opa1) that are involved in fusion andtwo proteins (drp1 and fis1) that are essential for division have been identified. The over-expression of fis in our system lead to extremely shortened mitochondria. These mito-chondria seemed unable to undergo further fusion, even with normal mitochondria, andtherefore no exchange of proteins took place. Comprising, these results stress the hypothe-sis of mitochondrial fusion as a rescue mechanism. Author Disclosure Block: K.B. Busch,None; M. Jendrach, None; J. Bereiter-Hahn, None.

POS-L90 BOARD #LB90Protein processing and plasma membrane targeting functions of the MultidrugResistance Protein (MRP)1 N-terminal membrane spanning domain. Chris J.Westlake, Susan P. C. Cole, Roger G. Deeley. Cancer Reseach Institute, Queen’sUniversity, Kingston, ON, Canada.The Multidrug Resistance Protein 1 (MRP1/ABCC1) confers resistance to chemothera-peutic agents, and exports organic anions conjugated to glutathione, glucuronate, and sul-fate. MRP1 is a member of the ‘C’ branch of ATP binding cassette (ABC) transporter fam-ily, which is comprised of ten MRPs, CFTR, and the sulfonylurea urea receptors (SUR). Inaddition to two membrane spanning domains (MSDs) typical of ABC transporters,MRP1-3, -6 and -7, and the SURs have an additional NH2-terminal MSD (MSD0).Unlike some topologically similar ABCC proteins, removal of MSD0 has minimal effecton MRP1 function, nor does it prevent trafficking to basolateral membranes. However, incomparison to the wild-type protein we find that twice as much of the NH2-terminal trun-cated MRP1 is detected intracellularly. Using real-time fluorescence studies, we demon-strate that MSD0-less MRP1 colocalizes with internalized transferrin in early/recyclingendosomes. These results suggest that MSD0 is important for retention in, or recycling to,the plasma membrane. We also show that MSD0 traffics independently to the cell surfaceand promotes plasma membrane localization of the core-region of MRP1 when coex-pressed. Contrary to our previous reports, which showed that the COOH-terminal regionwas not important for MRP1 trafficking, we have determined that removal of MSD0 andthe COOH-terminal region caused retention in the ER. These studies demonstrate thatMRP1 terminal regions contain redundant trafficking signals, which only become essentialwhen one or the other regions is mutated. Finally, chimeric proteins in which MRP1

MSD0 was exchanged for the analogous domains from MRP2 and MRP3 were not tar-geted to the plasma membrane in the absence of the COOH-terminal region. These dataexplain apparent differences in the trafficking requirement for MSD0 and the COOH-ter-minal region of MRP1 when compared with other ABCC proteins. Author DisclosureBlock: C.J. Westlake, None; S.P.C. Cole, None; R.G. Deeley, None.

POS-L91 BOARD #LB91Critical Amino Acid Residues Required for Sodium Bicarbonate Cotransport Mediatedby kNBC1. Rustam Azimov, Natalia Abuladze, Debra Newman, Alexander Pushkin, IraKurtz. UCLA, Los Angeles, CA, USA.We have previously reported a topological model of the electrogenic sodium bicarbonatecotransporter NBC1 (Tatishchev et al., 2003). In this model the cotransporter spans theplasma membrane 10 times with N- and C-termini localized intracellularly. An analysis ofconserved amino acid residues among mammalian and non-mammalian members of theBTS superfamily in both the transmembrane segments and intracellular/extracellular loopsprovided the basis for the mutagenesis approach taken in the present study. Using large-scalemutagenesis, acidic and basic amino acids putatively involved in ion binding/transport weremutated to neutral and/or oppositely charged amino acids. All mutant kNBC1 cotrans-porters were expressed in HEK293-T cells and the Na+-dependent HCO3- flux of themutants was measured using BCECF. Critical glutamate, aspartate, lysine, arginine and his-tidine residues in extracellular/intracellular loops and transmembrane segments were detect-ed that were essential for kNBC1-mediated Na+-dependent bicarbonate transport. In addi-tion, critical phenylalanine and serine residues in transmembrane segments were detected.Furthermore, several charged residues in the extracellular/intracellular loops and trans-membrane segments were shown to be essential for membrane targeting. A model ofkNBC1-mediated ion transport is presented. Author Disclosure Block: R. Azimov, None;N. Abuladze, None; D. Newman, None; A. Pushkin, None; I. Kurtz, None.

POS-L92 BOARD #LB92Regulation of the mechanical tension of cells by membrane microdomains.Pierre Sens, PhD1, Matthew S. Turner, PhD2. 1ESPCI, Paris, France, 2Department ofPhysics - Warwick University, Coventry, United Kingdom.Cells have develop means of regulating the mechanical tension of their plasma membranethrough a process called Surface Area Regulation (SAR). It has been proposed that somemembrane reservoir inside the cell is able to exchange membrane area with the plasmamembrane to release some of its tension. We present a model that shows that membranedomains with associated membrane proteins (i.e. rafts or caveolae) can constitute such areservoir, by forming micro-undulations of the plasma membrane that flatten as tension

increases. Our model shows that largeenough membrane domains (50-100nm in size) invaginate from a low-ten-sion membrane. A mechanical pertur-bation of the cell (pulling a membranetether from the plasma membrane forinstance) increases the membrane ten-sion up to a value where some invagi-nations flatten. The coexistencebetween invaginated and flat domainsallow the cell to maintain its mem-brane tension to a constant value, up

to a level of perturbation for which the reservoir is emptied.We propose that many important insights on the formation and stability of large mem-brane domains can be obtained by studying the variation of the cell membrane tensionupon mechanical perturbation. Author Disclosure Block: P. sens, None; M.S. Turner, None.

POS-L93 BOARD #LB93Interaction of Phospholemman with Proteins of Cardiac Sarcolemma. Paul Perring1,Alexander Ivanov, Ph.D.2,1. 1Medical College of Ohio, Toledo, OH, USA,2Massachusetts General Hospital/Harvard Medical School, Boston, MA, USA.Phospholemman (PLM) is a member of the family of FXYD proteins. Although it was dis-covered as a major substrate of PKA and PKC in cardiac sarcolemma, its role in the cardiacmyocyte remains enigmatic. It has been implicated in formation of ion channels, in mod-ulating Na,Ca-exchanger, and in being a tissue-specific modulatory subunit of Na,K-ATPase. For the last, it has many similarities to phospholamban regulation of SERCA. Herewe present evidence for two interaction partners, revealed by chemical cross-linking of puri-fied bovine cardiac sarcolemma with DSS and BS3 (agents that target primary amines, pre-dominantly lysines). PLM- Na,K-ATPase α-subunit is one of the major products formedby cross-linking. This suggests that PLM interacts directly with the ?-subunit in theoligomeric structure of Na,K-ATPase. PLM-caveolin-3 (a marker protein for caveolae inmyocytes) is another major cross-linking product. Both interactions were specific. Theywere detergent-stable because the crosslinks also formed in digitonin-solubilized sarcolem-ma. Cross-linking was not phosphorylation-dependent, since non-phosphorylated, PKA-phosphorylated, and PKC-phosphorylated forms of PLM served as partners in cross-link-ing with either the α-subunit of Na,K-ATPase or caveolin-3. Caveolae prepared by car-bonate extraction from heart tissue contained a significant amount of all forms of PLM.This points to the possible participation of PLM in signal transduction via Na,K-ATPase,since caveolae are considered to be domains of assembly of signal transducing cascades, andit has been suggested that the pool of Na,K-ATPase located in caveolae can perform a sig-naling function. Sponsored by Grants HL 36573 and HL 36271. Author Disclosure Block:P. Perring, None; A. Ivanov, None.

POS-L94 BOARD #LB94Gating of the Nav1.4 mutations causing hypokalemic periodic paralysis type II. AlexeyKuzmenkin, PhD1, Elza Amirgoulova2, Frank Lehmann-Horn, MD, PhD1, KarinJurkat-Rott, MD, PhD1. 1Dept of Applied Physiology, University of Ulm, Ulm,Germany, 2Dept of Biophysics, University of Ulm, Ulm, Germany.Hypokalemic periodic paralysis (HypoPP) is a dominantly inherited neurologic disorder

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characterized by episodes of muscle weakness and a decreased potassium serum level dur-ing paralytic attacks. Previous genetic studies revealed mutations in the voltage-gated calci-um channel alpha1-subunit (CACNA1S gene, HypoPP type I) and skeletal muscle sodi-um channel alpha-subunit (SCN4A gene, HypoPP type II) in affected families. Whereasthe pathophysiology of the HypoPP type I still remains unclear, the HypoPP type II muta-tions were found to decrease current density and to cause stabilization of the fast and slowinactivated states (Kuzmenkin et al., 2002; Brain 125: 835-43). Since all the HypoPP type2 mutants are located in the voltage sensor of the second domain (D2/S4) and are replace-ments of the positively charged arginines by the uncharged amino acids, it was of great inter-est to elucidate how the mutant channels behave on the gating level. We heterologouslyexpressed R669H, R672H, R672G and WT Nav1.4 channels in a tsA-201 cell line andstudied them by means of the whole-cell patch-clamp technique. Our results suggest dis-tortion of the Nav1.4 activation process caused by the HypoPP mutations and give a newinsight into pathophysiology of the disease. Furthermore, the results contribute to under-standing of the Nav1.4 gating in general. Author Disclosure Block: A. Kuzmenkin, None; E.Amirgoulova, None; F. Lehmann-Horn, None; K. Jurkat-Rott, None.

POS-L95 BOARD #LB95Effect on potency of drugs using Li+ as a tracer for Na+ channels. Sikander Gill, DavidWicks, Rajwant Gill, Dong Liang. Aurora Bimed Inc, Vancouver, BC, Canada.Li+ is known to enter cells through Na+ channels at comparable permeability during patchclamp studies. Such studies support the use of Li+ as a tracer for Na+ channels and recep-tors in flux assays where Li+ was analyzed with the atomic absorption spectrophotometrybased Ion Channel Reader 8000. In these assays, cells are able to maintain a physiologicalmembrane potential when Li+ is used to replace Na+. Channel or receptor activation can beachieved with raising the extra-cellular KCl concentration or with specific agonists. Li+influx can be blocked or activated by specific blockers and activators respectively. In the pres-ent study, patch clamp was used to investigate the effect of using Li+ as a surrogate ion forNa+ in the bath solution. The comparison of potencies (IC50 or EC50) of specific blockersand activators of Na+ channels was carried out in the presence of Na+/Li+. Correlation withflux assays is also shown. Author Disclosure Block: S. Gill, Aurora Biomed Inc Employment(full or part-time); D. Wicks, Aurora Biomed Inc Employment (full or part-time); R. Gill,Aurora Biomed Inc Employment (full or part-time); D. Liang, Aurora Bimed Inc Ownershipor partnership.

POS-L96 BOARD #LB96Bupivacaine block of Kv channels suggests a PVP-hinge opening. Johanna Nilsson1,Viktor Luzhkov2, Johan Åqvist2, Peter Århem1. 1Karolinska Institutet, Stockholm,Sweden, 2Uppsala University, Uppsala, Sweden.The opening and closing of voltage-gated channels is at present a matter of controversy. Twomodels compete for acceptance. The glycine-bend model, mainly based on the crystalliza-tion of MthK, assumes that Kv channels open at a conserved glycine hinge of S6. The PVP-bend model, mainly based on the crystallization of KcsA and consequent mutational stud-ies of Shaker, assumes that the channels open at the proline rich region at the crossing of S6bundles.In the present study we have tried to relate these questions to the molecular mechanisms oflocal anesthetic block. We have analyzed the effects of bupivacaine on different types of wildtype and critically mutated Kv channels. Different Kv channel types were found to differ incapacity to close in bound state, suggesting that the binding site is located in the PVP regionand that the internal vestibule volume differs between channel types (with Kv1.5 beingmost narrow and least probable to close in bound state). To relate these findings to the dif-ferent opening models we performed autodocking and molecular dynamics simulations.We simulated bupivacaine binding to Kv1.5 homology models in the PVP-hinge and theglycine-hinge open state configuration. Estimating the binding free energy at the variousbinding sites we found that bupivacaine can bind to two regions in the internal vestibule ofthe PVP-bend model, but, surprisingly, not at all to the glycine-bend model.The findings thus support the PVP-bend opening model. We suggest that the differencebetween the two opening mechanisms depends on different evolutionary histories; theglycine-bend model applies to prokaryotic 2TM channels, while the PVP bend modelapplies to eukaryotic 6TM channels. This would explain the reported ten-fold smaller sin-gle channel conductance in Shaker than in bacterial 2TM channels. Author DisclosureBlock: J. Nilsson, None; V. Luzhkov, None; J. Åqvist, None; P. Århem, None.

POS-L97 BOARD #LB97High threshold K+ current increases gain by offsetting a frequency-dependent increasein low threshold K+ current. Fernando R. Fernandez, BSc, W. Hamish Mehaffey, BSc,Michael L. Molineux, BSc, Ray W. Turner, Phd. University of Calgary, Calgary, AB,Canada.High frequency firing neurons are found in numerous central systems, including the audi-tory brainstem, thalamus, hippocampus, and neocortex. The kinetics of high threshold K+

currents (IKHT) from the Kv3 subfamily has led to the proposal that these channels offsetcumulative Na+ current inactivation and stabilize tonic high frequency firing. All high fre-quency firing neurons examined to date, however, also express low threshold K+ currents(IKLT) that have slower kinetics and play an important role in setting the subthreshold andfiltering properties of the neuron. IKLT has also been shown to dampen excitability and istherefore likely to oppose high frequency firing. In this study we examined the role of IKHTin pyramidal cells of the electrosensory lobe of weakly electric fish, which are characterizedby high frequency firing, a very wide frequency range and high levels of IKHT. In particu-lar, we examined the mechanisms that allow IKHT to set the gain of the F-I relation by inter-acting with another low threshold K+ current. We found that IKHT increases the gain of theF-I relation and influences spike waveform almost exclusively in the high frequency firingrange. The frequency-dependence arises from IKHT influencing both the IKLT and Na+

currents. IKHT plays a significant role in stabilizing high frequency firing by preventing asteady state accumulation of IKLT that is as important as preventing Na+ current inactiva-tion. Author Disclosure Block: F.R. Fernandez, None; W. Mehaffey, None; M.L. Molineux,None; R.W. Turner, None.

POS-L98 BOARD #LB98A quantitative assessment of models for voltage-dependent gating of ion channels.Michael Grabe, PhD1, Harold Lecar, PhD2, Yuh-Nung Jan, PhD1, Lily Jan, PhD1.1UCSF, San Francisco, CA, USA, 2UC Berkeley, Berkeley, CA, USA.Voltage-gated ion channels open and close, or ̀ `gate”, in response to changes in membranepotential. The electric field across the membrane-protein complex exerts forces on chargedresidues driving the channel into different functional conformations as the membranepotential changes. In order to act with the greatest sensitivity, charged residues must be posi-tioned at key locations within or near the transmembrane region. This requires desolvatingcharged groups, a process which can be energetically prohibitive. While there is good agree-ment on which residues are involved in this process for voltage-activated potassium chan-nels, several different models of the sensor geometry and gating motions have been pro-posed. Here we utilize a novel approach of incorporating low-resolution structural infor-mation about the channel into a Poisson-Boltzmann calculation to determine solvation bar-rier energies and gating charge values associated with each model. The principal voltage-sensing helix, S4, is represented explicitly while all other regions are represented as feature-less, dielectric media with complex boundaries. From our calculations, we conclude that apure rotation of the S4 segment within the voltage-sensor is incapable of producing theobserved gating charge values, although this shortcoming can be partially remedied by firsttipping and then minimally translating the S4 helix. Models in which the S4 segment hassubstantial interaction with the low-dielectric environment of the membrane incur solva-tion energies of hundreds of kB T, and activation times based on these energies are ordersof magnitude slower than experimentally observed.Author Disclosure Block: M. Grabe, None; H. Lecar, None; Y. Jan, None; L. Jan, None.

POS-L99 BOARD #LB99Voltage Dependent Inactivation of Kv1.5 Caused by Internal Mg2+. Daniel C. H.Kwan, B.Sc1, Tom W. Claydon1, Shetuen Zhang2, David Fedida1, Steven J. Kehl1.1UBC, Vancouver, BC, Canada, 2University of Manitoba, Winnipeg, MB, Canada.Internal Mg2+ (Mg2+

i) is known to block a number of potassium channels. We have previ-ously shown that Mg2+

i induces voltage-dependent current decay at extreme depolarizationin Kv1.5. Here we show that the voltage-dependent decay is due to Mg2+

i block thatinduces P/C-type inactivation. Kv1.5 channels were expressed in HEK-293 or LM cells,and currents recorded under voltage clamp in whole-cell or inside-out patch configuration.Current traces were recorded in various [Mg2+]i from +40 to +200 mV from a holdingpotential of -80 mV. Current decay was fitted to double exponential functions (τfast andτslow). Mg2+

i enhanced both phases of current decay in a voltage- and concentration-dependent manner. With 10 mM Mg2+

i, τfast was reduced from 156±20 ms at +60 mV to5.4±1.1 ms at +140 mV and τslow was reduced from 2.2±0.2 to 0.2±0.01 s. In comparison,with 0 mM Mg2+

i, τfast and τslow were the same at +60 and +140 mV: τfast was 322±68 and206±47 ms and τslow was 3.1±0.6 and 3.3±0.9 s at +60 mV and +140 mV, respectively.Peak tail currents (at -50 mV) following 400 ms pulses to +200 mV were reduced to zerowith 10 mM Mg2+

i suggesting channel inactivation. In contrast, peak tail currents follow-ing 5 ms pulses to +200 mV, which allowed the fast, but not the slow current decay weresustained suggesting that the fast current decay is due to rapid channel block by Mg2+

i. Incontrast, increasing external K+ to 140 mM or the mutation R487V (equivalent to T449Vin Shaker), abolished the slow, but not the fast current decay suggesting that the slow decayis due to P/C-type inactivation. These results are consistent with open channel block ofKv1.5 by Mg2+

i that induces voltage-dependent P/C-type inactivation. Author DisclosureBlock: D.C.H. Kwan, None; T.W. Claydon, None; S. Zhang, None; D. Fedida, CardiomeStock options or bond holdings in a for-profit corporation or self-directed pension plan,Employment (full or part-time); S.J. Kehl, None.

POS-L100 BOARD #LB100Functional Analysis of a Re-engineered K+ Channel (maxi-K) for Development of anIon Channel-Based Hybrid Biosensor. Christopher R. Williams, Jun Zhang, RebeccaB. Price, Peter A. V. Anderson, Randolph S. Duran. University of Florida, Gainesville,FL, USA.The high-conductance voltage-activated potassium channel (maxi-K) is a robust, non-inac-tivating ion channel with known pharmacology and an inherent stochastic response tostimulus. These attributes make maxi-K an ideal candidate as the core component of an ionsensitive hybrid device. To optimize maxi-K for incorporation in a tethered-bilayer-baseddevice, the molecular structure of maxi-K channel has been modified. Using standardmolecular biology techniques, the large extramembranal domain (“Ca++-bowl”) wasremoved C-terminal to transmembrane helix S6 and a monomeric variant of the red fluo-rescent protein (mRFP1) was fused in frame at this position. A novel N-linked glycosyla-tion site was added between transmembrane helix S5 and the channel pore creating a cova-lent molecular adaptor for ricin sensitivity and possibly sensitivity to some viral particles.The properties of the mutant channels were monitored through heterologous expression inXenopus oocytes, and following reconstitution in unilamellar PS/PE liposomes. Fusion withmRFP1 enabled visualization of maxi-K expressed in Xenopus oocytes, COS-7 cells, andE.coli. Maxi-K re-engineered with the novel glycosylation site showed a distinct reductionin whole current amplitude following addition of ricin B-chain, but only after treatmentwith neuraminidase. The molecular structure of maxi-K was altered to reduce its size,increase its stability, and facilitate optical monitoring so as to optimize its use as a platformfor toxin and bio-particle detection. Further work will be directed towards characterizationof maxi-K incorporated in the tethered-bilayer-based hybrid device. Author DisclosureBlock: C.R. Williams, None; J. Zhang, None; R.B. Price, None; P.A.V. Anderson, None;R.S. Duran, None.

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POS-L101 BOARD #LB101Ethanol potentiation of ATP-gated currents on chimeric P2X2/3 receptors suggests aregion in purinergic receptors that is important for the action of ethanol. Andrei A.Kochegarov, PhD, Ronald L. Alkana, PharmD, PhD, Sacha T. Kuo, BS, Daryl L.Davies, PhD. University of Southern California, Los Angeles, CA, USA.P2X receptors (P2XRs) are cation-selective, ligand-gated ion channels (LGICs) activated byextracellular adenosine 5-triphosphate (ATP). Previously, we reported that ethanol inhibit-ed homomeric P2X2 and P2X4 receptors, but potentiated homomeric P2X3 receptors.Presently, the site(s) of ethanol action on P2XRs is unknown. The current study utilizes achimeric strategy that exploits the difference in ethanol action between P2X2Rs (ethanolinhibition) and P2X3Rs (ethanol potentiation) to begin to identify important regions forethanol action. We created a chimeric P2X2/3 receptor which contained transmembranedomain 1 (TM1) and N-terminal cytoplasmic part from P2X2 receptor (1-47) and extra-cellular loop and TM2 with C-terminal cytoplasmic part derived from P2X3 receptor (42-397). The effects of ATP and α,βmeATP (EC5-10) ± ethanol (5-200 mM) were measuredon Xenopus oocytes expressing rat wild type P2X2R and P2X3R or chimeric P2X2/3Rusing two-electrode voltage clamp (-70mV). We found that ethanol reversibly potentiatedboth ATP and α,βmeATP-gated currents in the chimeric P2X2/3R suggesting that animportant site of ethanol action is located in P2X3-derived region of the chimeric receptorin the region beyond TM1. Neither ATP or α,βmeATP caused desensitization in thechimeric P2X2/3R. This contrasts to the wildtype P2X3R which exhibits rapid desensiti-zation. This finding suggests that the region of the P2X3R including amino acids 1-47(region replaced in the chimeric P2X2/3R) plays an important role in fast desensitizationof P2X3Rs. Studies are currently underway to further refine the site of ethanol action bythe use of additional chimeric receptors. Support: NIAAA/NIH AA03972 (RLA),AA013890 (DLD), AA013922 (DLD) and the USC School of Pharmacy. AuthorDisclosure Block: A.A. Kochegarov, None; R.L. Alkana, None; S.T. Kuo, None; D.L. Davies,None.

POS-L102 BOARD #LB102DIFFERENTIAL POTENCY OF 2’-FLUORO-3’-(SUBSTITUTEDPHENYL)DESCHLORO EPIBATIDINE ANALOGS AS ANTAGONISTS FORalpha4beta2 AND alpha3beta4 NEURONAL nAChRs. Galya R. Abdrakhmanova1,F. Ivy Carroll2, M. Imad Damaj1, Billy R. Martin1. 1VCU, Richmond, VA, USA, 2Research Triangle Institute, Research Triangle Park, NC,USA.2’-fluoro-3’-(substituted phenyl)deschloroepibatidine analogs have been recently developedand shown to antagonize with high potencies, similar to or greater that those of mecamy-lamine, antinociceptive effects of nicotine in vivo (Carroll et al., J. Med. Chem., 2004).Interestingly, these epibatidine analogs were more effective in the tail-flick than in the hot-plate test, and, in contrast to mecamylamine, failed to antagonize nicotine-inducedhypothermia. In addition, these compounds possessed high affinity for α4β2, but no affin-ity for α7, nAChR binding. Patch-clamp technique was used to compare the effect of theanalogs in vitro on α4β2 and α3β4 subtypes of neuronal nAChRs stably expressed in SH-EP1and HEK 293 cell lines, respectively. Similarly to behavioral studies that revealed both3- and 4-nitrophenyl analogs being more potent than fluoro- or chlorophenyl analogs inthe hot plate test, the nitrophenyl analogs applied at 1μM inhibited ACh-induced currentsin α4β2 nAChRs by ~81 and ~65 %, while no substantial effect (q10% inhibition) wasobserved for both 3- or 4-fluorophenyl analogs neither at 1 nor at 10 μM on the samereceptor subtype, and only ~20% inhibition was induced by 4-chlorophenyl analog at10μM, similarly to its weakest antagonistic potency among these analogs in the hot platetest. In contrast when tested on α3β4 nAChRs, the most effective was 4-fluorophenyl ana-log (~87% inhibition at 10μM). 3-fluorophenyl and 3- or 4-chlorophenyl analogs inhibit-ed the ACh-induced current by 5 to 10% less at the same concentration, and, in contrastto their high efficacy on α4β2 receptors, the nitrophenyl analogs showed only 30 to 40%inhibition when applied at 10μM on α3β4 expressing cells. These results suggest specificsensitivity of α4β2 and α3β4 nAChRs to tested 2’-fluoro-3’-(substitutedphenyl)deschloroepibatidine analogs, that can be utilized in establishing nAChR subtype-specific antagonists. Author Disclosure Block: G.R. Abdrakhmanova, None; F.I. Carroll,None; M.I. Damaj, None; B.R. Martin, None.

POS-L103 BOARD #LB103Incorporation of a Fluorescent Unnatural Amino Acid into the Nicotinic Receptor.Rigoberto Pantoja, Ph.D., E. James Petersson, Mohammed I. Dibas, Ph.D., Dennis A.Dougherty, Ph.D., Henry A. Lester, Ph.D.Caltech, Pasadena, CA, USA.A fluorescent unnatural amino acid has been incorporated into the extracellular domain ofthe nicotinic acetylcholine receptor (nAChR) using nonsense suppression methodology. Analpha subunit with a Lys70TAG mutation was used to incorporate the Lys(NBD) fluores-cent unnatural amino acid. The alpha70 is in the main immunogenic region of the nAChR.Previous unnatural amino acid mutagenesis studies carried out in conjunction with biotin-streptavidin binding were used to demonstrate that alpha Lys70 is a surface exposed residue.This mutationally tolerant site was selected for incorporating the Lys(NBD) unnaturalamino acid, which is larger than natural amino acids. The nAChR proteins were expressedin Xenopus oocytes. The presence of surface-expressed nAChRs was confirmed by measur-ing macroscopic currents with a two-electrode voltage-clamp two days after injection. ThenAChR dose-response relationships were used to confirm phenotype characteristics.Fluorescent unnatural amino acids represent an attractive alternative for labeling ion chan-nel proteins with minimal structural perturbations. Motifs important for signaling path-ways will be influenced to a lesser extent as compared to fused green fluorescent proteins.Potential applications include higher resolution inter- and intra-molecular fluorescence res-onance energy transfer (FRET) experiments. Author Disclosure Block: R. Pantoja, None;E.J. Petersson, None; M.I. Dibas, None; D.A. Dougherty, None; H.A. Lester, None.

POS-L104 BOARD #LB104The Local Anesthetics Proadifen and Adiphenine Act at Different ConformationalStates of the Nicotinic Receptor. Guillermo F. Spitzmaul, PhD1, Fernanda A.Gumilar1, James P. Dilger, PhD2, Cecilia B. Bouzat, PhD1. 1Insituto de InvestigacionesBioquímicas, Bahía Blanca, Argentina, 2State University of New York at Stony Brook,Stony Brook, NY, USA.Local anesthetics act as noncompetitive inhibitors of the nicotinic acetylcholine receptor(AChR). Proadifen has been shown to increase desensitization of the AChR, but its mech-anism of action as well as that of its analog, adiphenine, have not been elucidated.Single-channel and macroscopic-current recordings from cells transfected with adult mus-cle AChR reveal that both drugs increase desensitization by acting at different conforma-tional states. At the single-channel level, both drugs decrease the frequency of openingevents, without significant changes in the mean open time. At high ACh concentrations,proadifen does not affect cluster duration. In contrast, adiphenine significantly decreasescluster duration, such reduction being about 90% at 20 µM. Proadifen decreases the peakof macroscopic currents activated by rapid application of ACh to outside-out patches(IC50= 23 μM), without changing the decay rate due to desensitization. The onset of inhi-bition by proadifen is slower than recovery (rates are 0.19±0.5/s and 0.34±0.07/s for onsetand recovery, respectively). Adiphenine increases the decay rate without changing the peakcurrent. For both drugs preincubation is necessary to exert their full actions, which are notdependent on membrane potential. Experiments with the αE262 mutant AChR (a sitelabeled by meproadifen) show no differences in inhibition by proadifen but less sensitivityto inhibition by adiphenine. In summary, our results show that these chemically-relateddrugs modulate different conformational states of the AChR. Proadifen desensitizes AChRsthat are in the closed state and adiphenine increases desensitization from the open state.Author Disclosure Block: G.F. Spitzmaul, None; F.A. Gumilar, None; J.P. Dilger, None;C.B. Bouzat, None.

POS-L105 BOARD #LB105Nanosecond Dynamics of the Acetylcholine Binding Protein: A Time-ResolvedFluorescence Anisotropy Approach. David A. Johnson, Ph.D.1, Ryan E. Hibbs2, PalmerTaylor, Ph.D.2. 1University of California, Riverside, Riverside, CA, USA, 2University ofCalifornia, San Diego, La Jolla, CA, USA.A growing body of evidence supports the idea that the acetylcholine binding protein(AChBP) from the stagnant pond snail Lymnaea stagnalis is structurally homologous to theextracellular domain of the nicotinic receptor (nAChR). Because large amounts of theAChBP but not the nAChR can be mutated and expressed, we have started to map thealpha-carbonyl backbone flexibility of the AChBP using a combination of cysteine substi-tution, fluorescence labeling, and time-resolved fluorescence anisotropy (TRFA). The longterm goal is to understand how ligands regulate the nAChR by assessing the effects ofcholinergic drugs on the conformational flexibility of the AChBP. So far, we have engi-neered and labeled five cysteines-substitution mutants with IAEDANS at positions K139,E157, N158, Y164, and T177. IAEDANS shows the greatest mobility attached to theT177C on the exposed surface of the lower C-loop. IAEDANS shows the least mobilityattached to the Y164C on the lower inner wall of the ACh binding pocket. Intermediatemobility of the IAEDANS was observed with the E157C and N158C to the right of theACh binding pocket and with the K139C mutant at inner left side of the ACh bindingpocket. (Supported by a U.S. Public Health Service grant R37-GM18360 and a PhRMApre doctoral fellowship). Author Disclosure Block: D.A. Johnson, None; R.E. Hibbs, None;P. Taylor, None.

POS-L106 BOARD #LB106Functional Contribution of Position L218 to Neuronal Nicotinic Alpha ThreeReceptor Subtypes. Daniel Caballero, B.S., Madeline Nieves-Cintrón, Ph.D., ManuelNavedo, Ph.D., José A. Lasalde-Dominicci, Ph.D.UPR-Río Piedras Campus, Río Piedras, PR, USA.Position L218 at transmembrane domain one (TMD1) is a highly conserved amino acidresidue among different neuronal α and β subunits, suggesting that this residue could beimportant for channel function. Site-directed mutagenesis and the two electrode voltageclamp technique were used to study the role of conserved position L218 on the TMD1 ofthe neuronal nAChR α3 subunit. The neuronal α3L218 position was substituted by fourdifferent amino acids (alanine, serine, phenylalanine and tyrosine) and the mutant α3 sub-unit was co-expressed with wild type β2 or β4 subunits in Xenopus laevis oocytes. The sub-stitution of L218 by alanine or serine produced a receptor loss in function with 8 and 15fold increases in the EC50 for ACh, respectively. The F and Y mutations did not affect thereceptor EC50 for ACh, nonetheless, the F substitution produced a reduction in the macro-scopic response elicited by ACh. We also examined how position L218 influenced the acti-vation of the α3 nAChRs subtypes by nicotine and 1,1-dimethyl-4-phenylpiperazinium(DMPP) and the sensitivity of the channel to competitive inhibition. Inhibition curves sug-gest that this position could affect agonist binding. The present results suggest that aminoacid volume on position L218 is critical for ion channel function and that structural alter-ations at this position could propagate to the agonist binding site. Author Disclosure Block:D. Caballero, None; M. Nieves-Cintrón, None; M. Navedo, None; J.A. Lasalde-Dominicci,None.

POS-L107 BOARD #LB107Movements of the S6 domain of CNGA1 channels during gating. Anilkumar V. Nair,MSc. Physics1, Monica Mazzolini, PhD2, Paolo Codega, Medical Biotechnology1,Alejandro Giorgetti, PhD3, Vincent Torre, PhD1. 1SISSA, Trieste, Italy, 2Istituto diBiofisica,Consiglio Nazionale delle Ricerche, Genova, Italy, 3Biocomputing Departmentof Biochemical Sciences University, Rome, Italy.All residues from Phe375 to Val424 were mutated one by one to a cysteine and modifica-tions induced by intracellular Cd2+ in presence of 1 mM cGMP and in its absence werestudied. The effect of other sulfhydryl reagents, such as MTSET, MTSES, MTSEA andCuP was analyzed on selected mutants. Cd2+ ions in the closed but not in the open stateblocked in an irreversible way many cysteine mutants in the final portion of the S6 domains

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from Asn400 to Arg422. The blocking effect of Cd2+ on mutant channels from F375C toS399C was less pronounced and was almost identical in the open and closed state. Ourresults suggest that the 3D structure of the KcsA is a good model for the orientation in spaceof the S6 domains of the CNGA1 channel in the closed state and that the 3D structure ofthe MthK channel is not an adequate model for the open state of the channel. A molecu-lar model of the 3D structure of the open state consistent with our data is obtained by usingas a template the HCN2 channel for the final portion of the S6 domain (approximatelyfrom Asn400 to Arg422) and the KcsA channel for the initial portion of the S6 domain(from Phe375 to Gly395). Author Disclosure Block: A.V. Nair, None; M. Mazzolini, None;P. Codega, None; A. Giorgetti, None; V. Torre, None.

POS-L108 BOARD #LB108How the pore of CNGA1 changes its conformation during gating. Anilkumar V. Nair,MSc. Physics1, Monica Mazzolini, PhD2, Katia Gamel, PhD3, Paolo Codega, MedicalBiotechnology1, Alejandro Giorgetti, PhD4, Vincent Torre, PhD1. 1SISSA, Trieste, Italy,2Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Genova, Italy, 3AnatomischesInstitut der Universität München, München, Germany, 4Biocomputing Department ofBiochemical Sciences University, Rome, Italy.The coupling between the pore walls, the P helix and the upper portion of the S6 trans-membrane domain of the CNGA1 channel from bovine rods was analyzed by electro-physiological experiments with several single and double mutant channels. Mutant channelT360C was potentiated by intracellular MTSES both in the open and closed state, butMTSES had no effect either on the mutant channel T359C or on the w.t. channel. Themutant L356C inactivated in the presence of a steady cGMP concentration, similarly towhat previously observed in mutants E363A,E363S and E363N ( Bucossi et al., 1996). Onthe contrary, the double mutant L356C & F380C did not inactivate. Also the mutantchannel T355C inactivated but in addition had a fast run down. The extensive analysis ofsingle and double mutant channels suggests that: i - the gate of CNGA1 channels is locat-ed in the pore itself; ii - the pore wall is coupled to the P-helix by interactions between sidechains of Glu 363 and Thr 355 and possibly also by a hydrophobic interaction between sidechain of Iso 361 and Leu 358; iii - the P helix and the upper part of S6 are coupled byhydrophobic interactions, in particular between side chains of Leu356 and Phe380 andpossibly by interactions between Asp 379 and Thr 353. These interactions strongly con-strain the molecular structure of the pore of CNGA1 channels and allow the constructionof a molecular model of channel gating initiated by an anticlockwise rotation of the S6 helix.Author Disclosure Block: A.V. Nair, None; M. Mazzolini, None; K. Gamel, None; P.Codega, None; A. Giorgetti, None; V. Torre, None.

POS-L109 BOARD #LB109Modulation of G-protein activated inward rectifier K+ currents by activation ofmetabotropic glutamate receptor in rat hippocampal CA1 neuron. Jong-Woo Sohn1,Hana Cho2, Suk-Ho Lee1, Won-Kyung Ho1. 1Seoul National University College ofMedicine, Seoul, Republic of Korea, 2Kangwon National University College of Medicine,Chuncheon, Republic of Korea.We have previously reported that G-protein activated inward rectifier potassium channels(GIRK) in cardiac myocytes are inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2)depletion induced by the activation of Gq-protein coupled receptor(GqPCR)s such as α1-adrenergic receptors. In the present study, we investigated the effect of group I metabotrop-ic glutamate receptor agonist DHPG (3,5-dihydroxyphenylglycine) on GIRK currents(IGIRK) recorded in rat hippocampal CA1 neurons. IGIRK was activated by baclofen (100μM), a GABAB receptor agonist. When baclofen was applied for 30 sec repetitively in a 3min interval, the peak amplitude of IGIRK in the second application of baclofen was 95.42± 10.15% of that in the first application. When 50 μM DHPG was applied prior to thesecond application of baclofen, the peak amplitude of IGIRK was 50.78 ± 12.84% of con-trol, indicating that DHPG reduced IGIRK. The degree of inhibition was not potentiatedwhen PIP2 replenishment was blocked by 100 μM wortmannin. Neomycin (100 μM), aphospholipase C inhibitor did not block the effect of DHPG. Neither GF109203X (100nM), a protein kinase C inhibitor nor staurosporine (1 μM), a broad spectrum proteinkinase inhibitor failed to block DHPG effect. Finally, AACOCF3 (50 μM), a phospholi-pase A2 inhibitor, significantly attenuated DHPG effect, suggesting the involvement ofphospholipase A2 / arachidonic acid pathways in IGIRK inhibiton by DHPG. Taken togeth-er, it was suggested that the mechanism of GIRK channel modulation by GqPCR differsamong different cell types. Author Disclosure Block: J. Sohn, None; H. Cho, None; S. Lee,None; W. Ho, None.

POS-L110 BOARD #LB110β-adrenergic receptor modulation of cardiac Na/Ca exchange in pig and dog myocytes.Shao-kui Wei, MD, John McCurley, MD, Matie Shou, MD, Mark Haigney, MD.Uniformed Services University, Bethesda, MD, USA.Whether the β-adrenergic receptor (β-AR) modulates Na/Ca exchanger (NCX) activity inmyocytes remains controversial. Significant β-AR modulation of NCX was reported in pig

and rat myocytes, but not confirmed in rabbit orguinea-pig myocytes. The Ca-activated Cl- cur-rent (ICa-Cl) and cAMP-dependent Cl- current(CFTR) are Ni+-sensitive and have similar rever-sal potentials to NCX, confounding measure-ments of NCX. In this study we further charac-terized the cAMP-sensitive current in swine andcanine myocytes. Methods: Left ventricularmyocytes were isolated from normal pig and doghearts. The Ni-sensitive current was measured byvoltage ramp. Results: In pig myocytes, blockadeof ICa-Cl (Niflumic acid 100 µmol/L) and CFTR(Glibenclamide 100 µmol/L) failed to reduce the

isoproterenol (ISO, 2 µmol/L)-activated current. The ISO-stimulated current was notaltered under free external Cl- but eliminated by removal of external Ca2+. In dog myocytes,

ISO and Okadaic acid (protein phosphatase inhibitor,1 µmol/L) increased current to anextent similar to pigs and was dependent on external Ca2+. Conclusions: β-AR stimulationof pig and dog myocytes activates a current that is most likely NCX and inconsistent withCa-activated Cl- current and cAMP-dependent CFTR. Author Disclosure Block: S. Wei,None; J. McCurley, None; M. Shou, None; M. Haigney, None.

POS-L111 BOARD #LB111Structure and Drug Inhibition Investigations of the Influenza A M2 Proton Channel.Philip A. Spearpoint1, Peter E. Czabotar, PhD2, Stephen R. Martin, PhD1, Patrick J.Collins, PhD1, Alan J. Hay, PhD1. 1National Institute for Medical Research UK,London, United Kingdom, 2The Walter and Eliza Hall Institute of Medical Research,Melbourne, Australia.The influenza A virus M2 protein is a minor component of the virus membrane, which,in its homotetrameric form, is a pH-activated, proton-selective channel and is the target ofthe anti-influenza drugs,amantadine and rimantadine.Optical spectroscopic measurements on Weybridge M2 performed in detergent micelleshave been used to study the effects of pH changes and the binding of anti-influenza drugs.Far-UV circular dichroism measurements showed that neither pH change nor drug bind-ing had any effect on the secondary structure of M2. However, lowering the pH from 8 to5 quenched the tryptophan fluorescence by ~ 30% and leads to a small red shift in the emis-sion maximum. Although the latter observation suggests that one or both of the trypto-phans in M2 are somewhat more solvent exposed at low pH, fluorescence quenching stud-ies at different pH values showed only small differences in accessibility to the neutralquencher acrylamide. The quenching of fluorescence at low pH is known to be associatedwith protonation of His-37 and the pH range in which this protonation occurs (apparentpK~5) is similar to that at which proton permeation through the channel occurs. pHdependent changes in the environment of Trp41 within the channel are consistent with thesuggested role for this residue within the proton translocation apparatus.The pH-induced quenching of tryptophan fluorescence was reversed by the addition ofanti-influenza drugs. Time-resolved measurements of this drug-induced reversal permittedthe determination of both association and dissociation rate constants (and therefore Kds)for the interaction of M2 with amantadine, rimantadine, and cyclooctylamine. AuthorDisclosure Block: P.A. Spearpoint, None; P.E. Czabotar, None; S.R. Martin, None; P.J.Collins, None; A.J. Hay, None.

POS-L112 BOARD #LB112Stochastic QM/MM Models for Proton Transport in Biological Systems: An EmpiricalValence Bond (EVB) Approach. Anton Burykin, Sonja Braun-Sand, Arieh Warshel.University of Southern California, Los Angeles, CA, USA.Proton transport (PT) plays a major role in biophysics in general and bioenergetics in par-ticular. In view of the crucial role of biological PT processes and recent controversies aboutways to simulate them [1], it is important to gain a quantitative molecular understandingof the factors that control such processes.While modeling actual time-dependent PT in biological systems one has to deal with upto microsecond time scales which are not accessible to QM/MM or even MD (which areanyhow not able to capture the PT process) methods. In order to overcome this problemwe have developed a new type of hybrid quantum/classical approach which combinesexplicit QM (EVB) representation of the chain of donor and acceptors and implicit repre-sentation (via the effective coordinates) of the environment (the rest of the protein/watersystem). The dynamics of the whole QM/MM system is described by stochastic (langevin)equations. This model takes into account the correct physics of proton charge delocaliza-tion and the reorganization of solvent polar groups during the PT process.The description of QM/MM langevin dynamics method is given and several applicationsto biological systems (PT in Gramicidin A channel [2] and Carbonic Anhydrase [3]) arepresented.References1. Yarnell A. Blockade in the Cell’s Water Way, Chemical and Engineering News, 82:42-44 (2004)2. Braun-Sand, S, Burykin, A, and Warshel, A, Realistic Simulation of Proton transportalong the Gramicidin channel: demonstrating the importance of solvation effects. J. Phys.Chem. B (in press).3. Braun-Sand S., Strajbl M., Warshel A., Studies of Proton Translocations in BiologicalSystems: Simulating Proton Transport in Carbonic Anhydrase by EVB Based ModelsBiophys. J. 87: 2221-2239 (2004).Author Disclosure Block: A. Burykin, None; S. Braun-Sand, None; A. Warshel, None.

POS-L113 BOARD #LB113A Single-cell Electroporation Chip. Adrian Y. Lau, Michelle Khine, Cristian Ionescu-Zanetti, Jeonggi Seo, Luke P. Lee. University of California, Berkeley, Berkeley, CA, USA.Increasing the cell membrane’s permeability can be accomplished via single cell electropo-ration. Polar substances that cannot otherwise permeate the plasma membrane (such asdyes, drugs, DNA, proteins, peptides, and amino acids) can thus be introduced into thecell. We developed a polymeric chip that can selectively immobilize and locally electropo-rate single cells. This easy-to-use chip focuses the electric field and thus eliminating the needto manipulate electrodes or glass pipettes. We demonstrated the effectiveness of our devicedesign by electroporating Hela cells using low applied voltages (<1V). We found the aver-age transmembrane potential required for electroporation of Hela cells to be 0.51V+ 0.13.Membrane permeation is assessed electrically by measuring characteristic ‘jumps’ in currentthat correspond to drops in cell resistance, and microscopically by recording either theescape of cytoplasmic dye Calcein AM or the entrance of Trypan blue stain. We also foundthat this device enables multiple single cell trapping and electroporation under standardizedcondition, allowing extensive statistically study of electroporation biophysics. AuthorDisclosure Block: A.Y. Lau, None; M. Khine, None; C. Ionescu-Zanetti, None; J. Seo, None;L.P. Lee, None.

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POS-L114 BOARD #LB114biophysical analysis of neural correlates to c. elegans thermotaxis. Damon A. Clark1,Keng-hui Lin, PhD1,2, Christopher V. Gabel, PhD1, Samuel H.-K. Chung1, StuartMilstein, PhD3, Eric Mazur, PhD1, Aravinthan Samuel1. 1Harvard University, Cambridge, MA, USA, 2Institute of Physics, Academia Sinica,Taipei, Taiwan Republic of China, 3Dana-Farber Cancer Institute, Boston, MA, USA.The simple and invariant neural circuit of the nematode C. elegans is well-suited for thestudy of the neural encoding of behavior. We have been studying worm thermotacticbehavior, in which animals navigate temperature gradients by crawling towards a preferredtemperature. First, we developed a new quantitative assay of thermotactic behavior in whichwe expose worms in a droplet to defined temporal thermal stimuli and monitor thrashingpatterns of individual worms. This assay measures the amplitude and dynamics of thermo-tactic stimulus-response relationships. Second, by manipulating droplet chemistry we findthat thermotactic decision-making involves integration of both chemosensory and ther-mosensory inputs, which implies a certain overlapping of neural circuits. Third, we use fem-tosecond pulsed laser surgery to sever individual dendrites of the sensory neurons of wild-type and mutant in order to localize specific sensory events to individual neural structures.Our work is allowing us to map behavioral pathways to their underlying neural circuitrywith new experimental assays that should have wider applications. Author Disclosure Block:D.A. Clark, None; K. Lin, None; C.V. Gabel, None; S.H. Chung, None; S. Milstein, None;E. Mazur, None; A. Samuel, None.

POS-L115 BOARD #LB115Intracellular Ion Transport in the Outer Hair Cell: A Finite Element Approach.William J. Triffo, Robert M. Raphael. Rice University, Houston, TX, USA.The outer hair cell (OHC) of the mammalian cochlea possesses the remarkable ability toutilize changes in transmembrane potential to drive cellular length changes. This phenom-enon, known as electromotility, is responsible for the exquisite sensitivity and frequency dis-crimination evident in the mechanical response of the organ of Corti. Our understandingof peripheral auditory physiology and subsequent direction for treatment of deafness aretherefore correlated to our comprehension of the physical basis of OHC operation. Due tothe coupling between transmembrane potential and axial deformation in the OHC, inter-pretation of in vitro experimental results and understanding of in vivo OHC physiologydepend on an accurate characterization of the development of the potential field over spaceand time. Because the charge carriers are ions in solution, correct depiction of the potentialnecessitates examination of intracellular ion transport. Previous studies of OHC electro-physiology have been limited to the one-dimensional (1D) case. We are currently imple-menting 3D finite element (FE) codes to apply conventional continuum theories of iontransport to the OHC, such as the Poisson-Nernst-Planck (PNP) description of electrodif-fusion. This implementation frees us from the restrictions of symmetry assumptionsrequired of lower dimension approximations. We discuss our progress in the application ofthese codes to the analysis of the whole-cell patch clamp setup, a technique commonly usedin OHC experiments. Author Disclosure Block: W.J. Triffo, None; R.M. Raphael, None.

POS-L116 BOARD #LB116Cardiac Sodium Channelopathies Probed by Dynamic Action Potential Clamp. GézaBerecki, Jan G. Zegers, Arie O. Verkerk, Ronald Wilders, Antoni C. G. van Ginneken.Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.Mutations in the cardiac sodium channel gene (SCN5A) are thought to be responsible fora number of congenital cardiac electrical diseases, like a subtype of the long QT syndrome,LQTS3, and the Brugada syndrome. We have recently introduced the dynamic actionpotential clamp (dAPC) technique (Biophys. J. 2005; 88:in press), which allows rapid andunambiguous determination of the effects of an ion channel mutation on the cardiac ven-tricular action potential (AP), without making assumptions with regard to the kinetic prop-erties of the channels.Here, we use the dAPC approach to effectively replace the native sodium current (INa) of aventricular myocyte or cell model with INa recorded at 35±1°C from a transfected HEK-293 cell that is voltage clamped by the free-running AP of the ventricular cell. To this end,the native INa is pharmacologically blocked (or set to zero in case of a model cell) and HEK-293 cell background currents are removed by real-time digital subtraction. When wild-type(WT) INa is added to the net membrane current of the ventricular cell, the resulting APshould be considered as “normal”, whereas a mutant (Y1795C, A1330P, ΔKPQ, and1795insD) INa should cause distortion of the AP. The resulting APs were compared withrespect to upstroke velocity, amplitude, duration (APD), rate-dependent alterations ofAPD, and pause-dependent restitution properties. Our results reveal not only frequency-dependent AP prolongation but also specific kinetic features of sodium channels, using con-ditions under which they normally function (i.e., by letting them generate and shape theventricular AP). Supported by Netherlands Heart Foundation Grant 2001B155. AuthorDisclosure Block: G. Berecki, None; J.G. Zegers, None; A.O. Verkerk, None; R. Wilders,None; A.C.G. van Ginneken, None.

POS-L117 BOARD #LB117Activation of hERG current by RO4606764. Liudmila Polonchuk, Rolf Osterwalder,Peter Mohr. F. Hoffmann-La Roche Ltd., Basel, Switzerland.During drug development new chemical entities are these days routinely tested for theirability to block the repolarizing K+ current through the hERG encoded cardiac ion chan-nels. Blockade of the hERG channels is known to be causatively involved in triggering ofcardiac arrhythmias. While the question about the functional importance of the hERGchannel blockade is extensively studied, the electrophysiological importance and proar-rhythmic relevance of hERG channel activation have not been thoroughly investigated sofar. The objective of this study was to characterize the electrophysiological effects ofRO4606764, a molecule that activates hERG current. Patch clamp experiments withCHO cells stably transfected with hERG gene demonstrated that RO4606764 significant-ly increases the hERG current in a concentration-dependent manner over the range of 1-100 uM. Similarly, RO4606764 amplifies the hERG current amplitude without significanteffects on the current kinetics. RO4606764 also induced a reduction of action potential

duration in isolated rabbit Purkinje fibers in the same concentration range. Thus, this com-pound may be useful to study the physiological role of the hERG channel activation in vitroand in vivo. Author Disclosure Block: L. Polonchuk, None; R. Osterwalder, None; P. Mohr,None.

POS-L118 BOARD #LB118Gender Differences in Electrophysiological Properties of Guinea Pig Heart.Judith Brouillette, Marie-Andrée Lupien, Céline Fiset. Montreal Heart Institute, Facultyof Pharmacy, University of Montreal, Montreal, PQ, Canada.Gender difference in cardiac repolarization have been observed in different species.However, little is known about sex differences in guinea pig ventricular repolarization. Here,we studied electrophysiological properties of adult male and female guinea pig ventricles.Using voltage-clamp techniques, we found that L-type Ca2+ currents (ICaL) recorded infemales myocytes (at 0 mV, -9.3 ± 0.5 pA/pF, n=16) were 20% smaller than in males (-11.2± 0.6 pA/pF, n=18, p=0.02). Next, we compared the density of the inward rectifier K+ cur-rent (IK1) and observed that the inward portion of IK1 was similar in both groups (at -110mV (pA/pF), males: -54.2 ± 4.1, n=11; females: -59.3 ± 3.7, n=14, p=NS) whereas the out-ward portion of IK1 was 22% larger in male myocytes (at -60 mV (pA/pF), males: 7.7 ±0.5; females: 6.3 ± 0.4, p=0.02). We then compared action potential durations (APD) andcorrected QT intervals (QTc) and found no sex-linked differences in these parameters(APD90 at 4 Hz (ms), males: 164 ± 4, n=34; females: 162 ± 5; n=29, p=NS) (QTc (ms),males: 227 ± 6, n=10; females: 241 ± 8, n=10, p=NS). To test whether the differences weobserved in both ICaL and IK1 could diminish each other effects on APD, we applied cad-mium (Cd2+) and barium (Ba2+) (100 µM each) to block ICaL and IK1. In presence of bothblockers, APD90 were similar between male (151 ± 9 ms, n=14) and female (149 ± 10 ms;n=11) myocytes and, of note, APD90 were also similar to those recorded under control con-ditions. Our findings suggest that the sex differences we documented in ICaL and IK1 couldpossibly counterbalance each other and contribute to explain the fact that male and femaleguinea pig ventricles display similar APD90 and QTc interval. Author Disclosure Block: J.Brouillette, None; M. Lupien, None; C. Fiset, None.

POS-L119 BOARD #LB119Conformational Changes of Troponin C within the Thin Filaments Detected byNeutron Scattering. Fumiko Matsumoto1,2, Kouji Makino2, Kayo Maeda2, HeikoPatzelt3, Yuichiro Maéda2, Satoru Fujiwara1. 1JAERI, Ibaraki-ken, Japan, 2RIKENHarima Institute, Hyogo-ken, Japan, 3Sultan Qaboos University, Sultan, Oman.Regulation of skeletal and cardiac muscle contraction is associated with structural changesof the thin filament-based proteins, troponin consisting of three subunits (TnC, TnI, andTnT), tropomyosin, and actin, triggered by Ca2+-binding to TnC. Knowledge of in situstructures of these proteins is indispensable for elucidating the molecular mechanism of thisCa2+-sensitive regulation. Here the in situ structure of TnC within the thin filaments wasinvestigated with neutron scattering, combined with selective deuteration and the contrastmatching technique. Deuterated TnC (dTnC) was first prepared, this dTnC was thenreconstituted into the native thin filaments, and finally neutron scattering patterns of thesereconstituted thin filaments containing dTnC were measured under the condition wherenon-deuterated components were rendered ‘invisible’ to neutrons. The obtained scatteringcurves arising only from dTnC showed distinct difference in the absence and presence ofCa2+. These curves were analyzed by model calculations using the Monte Carlo method, inwhich inter-dTnC interference was explicitly taken into consideration. The results of themodel calculations suggested that the thin filaments in the presence of Ca2+ are more dis-ordered locally than in the absence of Ca2+. More importantly, an in situ conformationalchange of TnC within the thin filament was detected for the first time. The model calcu-lation showed that the in situ radius of gyration of TnC in the absence of Ca2+ was 22 Å(99% confidence limits between 21 Å and 23 Å), which is consistent with the extendeddumbbell structure, while that in the presence of Ca2+ was 24 Å (99% confidence limitsbetween 23 Å and 26 Å), indicating that TnC became elongated by about 4 Å. AuthorDisclosure Block: F. Matsumoto, None; K. Makino, None; K. Maeda, None; H. Patzelt,None; Y. Maéda, None; S. Fujiwara, None.

POS-L120 BOARD #LB120Individually Addressable Microfluidic Patch Clamp Array System Using Time DivisionMultiplexing for High-Throughput Ion Channel Screening. Michael T. Mueller,Michelle Khine, Cristian Ionescu-Zanetti, Ph.D, Niraj Patel, Luke P. Lee, Ph.D. UCBerkeley, Berkeley, CA, USA.We applied time division multiplexing as a means to reduce the amplifier hardware require-ment for a high-throughput patch clamp electrophysiology system. We have designed,developed, and tested an integrated patch clamp array of four channels requiring only a sin-gle amplifier. Our complete system consists of custom designed hardware (a PCB interfacedwith microfluidic channels created in PDMS), and recording software created withLabVIEW. While the patch clamp is the “gold standard” assay for targeting ion channels,and provides superior data quality, it is inherently low-throughput and requires tediousmanual labor that makes it economically unsuitable for applications in high throughput ionchannel screening. Automation without the sacrifice of data quality is a prerequisite for apatch clamp array for ion channel screening in drug discovery.Our device addresses these problems by creating an array of microfluidic patch-clampdevices using polydimethylsiloxane (PDMS) elastomer connected with Ag/AgCl electrodesto a printed circuit board. The printed circuit board contains low-leakage multiplexing cir-cuitry to individually address and read data from patch clamp channels. Electrophysiologydata is reported to a computer through a National Instruments data acquisition system run-ning LabVIEW. The printed circuit board replaces tedious hand labor with computer con-trol of fluidic channel recording. We trapped multiple HeLa cells and achieved resistancemeasurements of nearly 100 MΩ. The design is robust and highly scalable, demonstratingthe feasibility of an automated patch-clamp array for high-throughput ion channel screen-ing, as well as a flexible electro-fluidic interface platform for cellular bioassay systems orbiofluidic application specific integrated circuits. Author Disclosure Block: M.T. Mueller,None; M. Khine, None; C. Ionescu-Zanetti, None; N. Patel, None; L.P. Lee, None.

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POS-L121 BOARD #LB121Serotonin causes contraction of intrapulmonary arteries involving Rho-RhoA and pro-tein kinase C signaling and store operated calcium channels. Zhigang Hong, MD,Ph.D, Andrew Smith III, MD, Dehong Li, MD, Anthony Varghese, Ph.D, Daniel P.Nelson, BS, E Kenneth Weir, MD. Dept. of Medicine, VA Medical Center andUniversity of Minnesota, Minneapolis, MN, USA.5-hydroxytryptamine (5-HT) is a potent pulmonary vasoconstrictor and plays an impor-tant role in the pathogenesis of pulmonary artery hypertension. Using the patch clamp tech-nique, we found that neither 5-HT (1 μM n=5, 10 μM n=5, 100 μM n=5) nor bone mor-phogenetic protein 2 (30 ng/ml n=8) had any effect on potassium current and membranepotential in freshly isolated rat resistance pulmonary artery smooth muscle cells (PASMC).10 μM 5-HT induced Ca++ current in resistance PASMC, which was sensitive to 1 mMNi (n=6). In denuded intrapulmonary artery rings, 5-HT caused dose-dependent contrac-tion with maximal response at a concentration of about 30 μM. 30 μM 2-APB, 10 μMSKF 96365, or 30 μM cyclopiazonic acid significantly inhibited 10 μM 5-HT-induced PAcontraction (52 ±13% n=6, 96 ±4% n=5, 82 ±9% n=3). Omitting outside bath calciumsignificantly inhibited 10 μM 5-HT-induced contraction (80 ±5%, n=6). Changing bathCa++ from 0 to 2 mM after 10 μM 5-HT and 3 μM nifedipine pretreatment caused sig-nificant contraction indicating involvement of store-operated calcium currents (SOCC). 3μM Y-27632 (Rho-kinase inhibitor) inhibited 85 ±3% of 10 μM 5-HT-induced contrac-tion. 50-300 nM H89 (PKA inhibition) had no effect, but 10 μM H89 (PKC inhibition)completed inhibited 10 μM 5-HT-induced contraction. 30 μM genistein and 100 μM tyr-phostin 25 (tyrosine kinase inhibition) also significantly inhibited 10 μM 5-HT-inducedcontraction (70 ±3% n=5, 22 ±4% n=2). Our results suggest that serotonin can activateSOCC and thus cause PA ring contraction. This may be associated with activity of Rho-RhoA and protein kinase C. Supported by National Heart Lung and Blood Institute GrantRO1 HL-65322. Author Disclosure Block: Z. Hong, None; A. Smith III, None; D. Li,None; A. Varghese, None; D.P. Nelson, None; E.K. Weir, None.

POS-L122 BOARD #LB122Correction of the Primary Defect in Dystrophin Deficient Single Cardiac Myocytes byMembrane Sealant Poloxamer 188. So-ichiro Yasuda, Elizabeth Favre, Joseph M.Metzger. University of Michigan, Ann Arbor, MI, USA.Dystrophin deficiency causes skeletal muscle disease that often involves cardiomyopathy,but it is unknown whether dystrophin deficiency directly causes altered force transmissionand/or membrane fragility in cardiac myocytes. Thus we measured tension and intracellu-lar calcium concentrations ([Ca]i) of isolated membrane intact myocytes using a uniquemicro-carbon fiber technique. Passive tension and [Ca]i of mdx cardiac myocytes were sig-nificantly greater than in control (C57BL/10) myocytes (passive tension, 6.3 ± 0.78 vs 4.1± 0.31 mN/mm2, mdx vs control, mean ± SEM, P<0.05, and [Ca]i, 207 ± 19.9 vs 129 ±10.2 nM, P<0.05, n=7 cells each) at 2.1 μm sarcomere length (SL). With further stretch(>2.15 μm SL), mdx myocytes became unstable with contracture, followed by cell deathassociated with increased [Ca]i (>1 μM). Treatment with 0.15mM Poloxamer 188 (P188),a compound that seals injured cell membranes, reduced the passive tension (4.2 ± 0.27mN/mm2, n=7) and [Ca]i (110 ± 10.5 nM, n=7) in mdx cardiac myocytes at 2.1 μm SL;and the treated mdx myocytes were stable at 2.15 - 2.2 μm SL. To assess the effect of stressduring active contraction, myocytes were stretched during twitch contractions. With 22-25% stretch of the resting myocyte length during a twitch, subsequent isometric twitch forcedecreased to 50% or less of the twitch force before stretch in mdx cardiac myocytes, whileno significant attenuation of twitch force was observed after the same extent of stretch incontrols. After treatment with P188, the post-stress twitch force of mdx cardiac myocyteswas partially recovered. These findings establish the primary defect in dystrophin-deficientcardiac myocytes as stretch-mediated contractile dysfunction and myocyte cell death, withincreased sarcolemma fragility, that can be prevented by membrane sealing compoundP188. Author Disclosure Block: S. Yasuda, None; E. Favre, None; J.M. Metzger, None.

POS-L123 BOARD #LB123Dynamics of Myosin Driven Skeletal Muscle Contraction. Ganhui Lan, Sean Sun.Johns Hopkins University, Baltimore, MD, USA.Skeletal muscle contraction is a canonical example of motor driven force generation.Despite the long history of research in this topic, a mechanistic explanation of the collectivemyosin force generation is lacking. We present a theoretical model of muscle contractionbased on the conformational movements of individual myosins and experimentally meas-ured chemical rate constants. Detailed mechanics of the myosin motor and the geometryof the sarcomere are taken into account. Two possible scenarios of force generation areexamined. We find only one of the scenarios can give rise to a plausible contraction mech-anism. We propose that the synchrony in muscle contraction is due to a force dependentADP release step. Computational results of a half sarcomere with 150 myosin heads canexplain the experimentally measured force-velocity relationship and efficiency data. We pre-dict that the number of working myosin motors increases as the load force is increased, thusshowing synchrony among myosin motors during muscle contraction. We also find thattitin molecules anchoring the thick filament are passive force generators in assisting musclecontraction. Author Disclosure Block: G. Lan, None; S. Sun, None.

POS-L124 BOARD #LB124Stochastic movements of muscle myosin; from single head to filament. Seiji Esaki1,Yoshiharu Ishii2, Toshio Yanagida1,2. 1Osaka University, Osaka, Japan, 2Formation of softnano-machines, CREST, Osaka, Japan.A recent study with single molecule measurements has reported that muscle myosin, amolecular motor, generates stochastic and multiple steps along an actin filament during thehydrolysis of a single ATP molecule. Here we proposed a model arguing the thermally-driv-en movements of a myosin head and extended the model to the movements of multipleheads and a filament.First, the movements of individual myosin molecules have been simulated based onBrownian movement along periodic and asymmetric potentials created by the interactionbetween myosin and an actin filament using the Langevin equation. Brownian movement

was biased to one direction by modulating the potential spatially or temporally. This modelcould successfully explain the movements of a single myosin head measured experimental-ly.Second, in attempt to show the cooperative features for the movement of multi-headedmotors, we traced the movement of dual- and multi-headed motors on connectingBrownian particles under the modulated potentials. The directionality of the movementbecame higher when each heads were connected by a linear spring with the moderate elas-ticity. Thus coordination of heads could bias the movement more effectively.Finally, we examined the possibility that the cooperation of the multiple heads movedlonger during hydrolysis of single ATP molecules, as reported that an actual actin filamentslides longer than 60 nm. Our simulation showed the multiple cooperating heads under-went stochastic steps to generate a long (> 60 nm) sliding distance per ATP between actinand myosin filaments while a myosin head moves at most 30 nm per ATP. Our model sup-ports the movement is loosely coupled to the ATPase cycle as observed in muscle. AuthorDisclosure Block: S. Esaki, None; Y. Ishii, None; T. Yanagida, None.

POS-L125 BOARD #LB125STRUCTURAL EFFECTS OF PKC-PHOSPHORYLATION OF THE N-DOMAIN OF CARDIAC TROPONIN I BOUND TO THE C-LOBE OF CAR-DIAC TROPONIN C. Natosha L. Finley, Paul R. Rosevear, PhD. University of Cincinnati, Cincinnati, OH, USA.PKC mediated phosphorylation of thin filament proteins diminishes contractility poten-tially leading to heart failure. Phosphorylation at Ser 43 and 45 of cardiac troponin Idecreases maximal actomyosin MgATPase activity, Ca2+ -sensitivity, and cross-bridge bind-ing to the thin filament. Troponin I is a member of the troponin complex, a ternary assem-bly of proteins responsible for the Ca2+ -sensitivity of muscle contraction. The cardiac tro-ponin complex contains the Ca2+ -binding protein, troponin C (cTnC), the inhibitorycomponent troponin I (cTnI), and the tropomyosin -binding protein troponin T (cTnT).While the physiological consequences of PKC phosphorylation of cTnI at Ser 43/45 areunderstood, the molecular mechanism responsible for these changes is unknown. Mutationof the Ser 43/45 to Asp, thereby introducing negative charges at positions 43 and 45 ofcTnI, has been shown in vitro and in vivo to mimic the physiological responses of phos-phorylation. Multidimensional heteronuclear NMR was used to study the structural con-sequences of the PKC phosphorylation mimetic of cTnI, cTnI(33-80; S43D, S45D)(NcTnI(DD), on the C -domain of cTnC (CcTnC), containing the Ca2+/Mg2+ -depend-ent interaction sites. We present a high-resolution NMR structure of the complex ofCcTnC bound to NcTnI(DD) and compare it with our previously determined structurefor CcTnC bound to NcTnI. Conformational perturbations were detected within theCa2+/Mg2+ binding loops III and IV and helix G of CcTnC. Additionally, the introduc-tion of negative charges in the N-terminus stabilized the helical content of NcTnI(DD).These data support previous studies of CcTnC bound to NcTnI(DD) identifying confor-mational alterations in helices F and G and the Ca2+/Mg2+ -binding loops. Taken togeth-er, our studies support a mechanism by which PKC phosphorylation of cTnI exerts itsfunctional effect through the Ca2+/Mg2+ interaction site. Author Disclosure Block: N.L.Finley, None; P.R. Rosevear, None.

POS-L126 BOARD #LB126Fesselin inhibits actin-activated ATPase activity of myosin S1 in the presence andabsence of tropomyosin. Joseph M. Chalovich, Ph.D., Mechthild M. Schroeter. BrodySchool of Medicine at ECU, Greenville, NC, USA.Fesselin is an actin binding protein from smooth muscle that has properties indicative of arole in modulating cellular actin structure. Fesselin bundles actin filaments and acceleratesnucleation of actin polymerization. Acceleration of actin polymerization by fesselin is inhib-ited by Ca++-calmodulin. Because actin filaments serve both structural and contractile func-tions we also examined the effect of fesselin on activation of myosin S1 ATPase activity.Fesselin inhibited the activation of S1 catalyzed ATP hydrolysis in a concentration depend-ent manner in both the presence and absence of tropomyosin. This inhibition was unaf-fected by Ca++-calmodulin. In general, Ca++-calmodulin had a larger effect on the interac-tion of fesselin with G-actin than with F-actin. The inhibition of ATPase activity was dueto a large part to inhibition of the binding of myosin-S1 to actin during steady-state ATPhydrolysis. The competition of fesselin and S1 for actin binding was further shown by thedisplacement of fesselin from actin by rigor S1. S1 has a very high affinity for actin in theabsence of ATP and readily displaced bound fesselin. The competition of binding betweenfesselin and S1 suggests that both share a common binding site on actin. The observationthat Ca++-calmodulin selectively regulated the interaction of fesselin with G-actin supportsa role of fesselin in regulating cellular structure. Author Disclosure Block: J.M. Chalovich,None; M.M. Schroeter, None.

POS-L127 BOARD #LB127Transduction of the Scorpion Toxin Maurocalcine into Cells - Evidence that the ToxinCrosses the Plasma Membrane. Xavier Altafaj, Postdoc1, Eric Estève1, Kamel Mabrouk2,Alain Dupuis1, Sophia Smida-Rezgui1, Didier Grunwald1, Michel De Waard1, MichelRonjat, Investigator1. 1CEA-Grenoble, U607 INSERM, Grenoble, France, 2CNRS FRE2738, Faculté de Médecine Nord, Marseille, France.Maurocalcine (MCa) is a 33 amino acid residue peptide toxin isolated from the scorpionScorpio maurus palmatus. External application of MCa to cultured myotubes is known toproduce Ca2+ release from intracellular stores. MCa binds directly to the skeletal muscle iso-form of the ryanodine receptor, an intracellular channel target of the endoplasmic reticu-lum, and induces long-lasting channel openings in a mode of smaller conductance. Here,we investigated the way MCa proceeds to cross biological membranes in order to reach itstarget. A biotinylated derivative of MCa was produced (MCab) and complexed with a flu-orescent indicator (streptavidine-cyanine 3) in order to follow the cell penetration of thetoxin. The toxin complex efficiently penetrated in various cell types without requiring meta-bolic energy (low temperature) or implicating an endocytosis mechanism. MCa appearedto share the same features as the so-called Cell-Penetrating Peptides (CPP). Our results pro-vide evidence that MCa has the ability to act as a molecular carrier and to cross cell mem-

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branes in a rapid manner (1-2 min) making this toxin the first demonstrated example of ascorpion toxin that translocates into cells. Author Disclosure Block: X. Altafaj, None; E.Estève, None; K. Mabrouk, None; A. Dupuis, None; S. Smida-Rezgui, None; D.Grunwald, None; M. De Waard, None; M. Ronjat, None.

POS-L128 BOARD #LB128Effect of inorganic phosphate on excitation-contraction coupling in fast-twitch musclefibres of the rat. Derek S. Steele, BSc PhD, Adrian M. Duke, B.Sc. Ph.D. School ofBiomedical Sciences, Leeds, United Kingdom.Previous work suggests that accumulation of inorganic phosphate (Pi) within the cytosolmight contribute to altered SR Ca2+ release during skeletal muscle fatigue. This study inves-tigated the effects of cytoplasmic Pi on excitation-contraction (E-C) coupling in rat skeletalmuscle fibres. Single fast-twitch fibres (extensor digitorum longus) were mechanically skinnedunder oil. Thereafter, the t-tubules reseal and repolarise, allowing the physiological Ca2+

release process to be studied. Fibres were bathed in solutions mimicking the intracellularmilieu containing (mM) HDTA, 50; ATP, 8; Na+, 37; K+,126; Mg2+, 8.6 (1 mM freeMg2+); PCr, 10; EGTA, 0.03; HEPES, 90;Ca2+, 0.0001; Fluo-3, 0.02. pH 7.1. Musclecontraction was induced by electrical field stimulation, which elicits action potentials (APs)within the t-tubule system. Isometric force production and Ca2+ release from the SR(detected using fluo-3) were recorded simultaneously. Tetanic responses were induced bystimulation with 2 ms pulses (70 V) at 50 Hz for 250 ms. Following 1 minute exposure to40 mM Pi, AP-induced tetanic [Ca2+] was reduced by 13% ± 4.7 and the integral of theresponse by 25.7 ± 6.7. Peak force was reduced by 29.1 ± 4.6 and the integral by 38 ± 8.2response (n = 4, p<0.05). These results suggest that in the absence of other metabolicchanges, an increase in cytosolic [Pi] can inhibit SR Ca2+ release and force production. Thismay contribute to the development of fatigue in fast twitch fibres.Author Disclosure Block: D.S. Steele, None; A.M. Duke, None.

POS-L129 BOARD #LB129Microfabricated Devices for the Alignment of Self-Assembled Filamentous ProteinSystems. Linda S. Hirst, PhD. University of California, Santa Barbara, Santa Barbara,CA, USA.We report a technique for the alignment of filamentous self-assembled protein systems,such as F-actin bundles and microtubules, in a surface modified titanium or silicon micro-

fluidic device. Such Protein systems aredelicate and typically difficult to align asthey must be formed under carefullycontrolled solution conditions. Narrowchannels allow the protein system toassemble in-situ in a confined geometry,producing an aligned sample. Thedevice surfaces are modified via self-assembled monolayers to resist protein

adsorption. Biomolecular self-assembly can be investigated in a controlled fashion underdifferent molecular concentration gradients and conditions along the channel. This tech-nique produces highly aligned samples for structural studies carried out via x-ray scatteringand is also useful for mechanical studies of different filamentous assemblies. Use of a micro-fabricated device for protein studies is perfect for microscopy and also will allow for theincorporation of MEMS features into the device. Author Disclosure Block: L.S. Hirst, None.

POS-L130 BOARD #LB130The Binding of an N-Terminal Mutant of Cofilin to Actin. Deepak Chhabra,B.Med.Sc.(Hons), Brett Hambly, Ph.D., Neil Nosworthy, Ph.D., Cristobal dosRemedios, Ph.D. University of Sydney, Sydney, Australia.Cofilin is an essential modulator of actin in all eukaryotic cells. It regulates the state of actinassembly during cell movement and cell division, as well as having potential roles in geneexpression and cell signalling. An atomic model of the cofilin-actin complex has not yetbeen determined, although several regions of cofilin, particularly its N-terminal region, havebeen implicated in its interaction with subdomains 1 and 3 of the G-actin monomer. It islikely that cofilin has at least 2 sites capable of interacting with actin (see review by dosRemedios et al., for detailed report of the cofilin-actin interaction).Investigation of the actin-cofilin complex by fluorescence techniques have so far proved dif-ficult due to the inability of cofilin to be labelled by fluorophores. To overcome this hurdle,we have used molecular biology techniques to add 6 residues, including 2 cysteines, to theN-terminus of cofilin, as previously described by Nagaoka and colleagues (1995). This N-terminal mutant of cofilin (N-cys-cofilin) was subsequently labelled with fluorescene.We have demonstrated that labelled N-cys-cofilin differs markedly from the native protein,displaying altered actin binding properties. We also used circular dichroism spectroscopy toprobe any changes in secondary structural content to determine the effect of the mutation-insertion in the stability of the protein.dos Remedios, C.G., Chhabra, D., Kekic, M ., Dedova, I., Tsubakihara, M., Berry, D. andNosworthy, N.J. (2003) Actin Binding Proteins and Regulation of Cytoskeleton Filaments.Physiological Reviews 83: 433-473.Nagaoka, R., Kusano, K., Abe, H. and Obinata, T. (1995) Effects of cofilin on actin fila-mentous structures in cultured muscle cells. Journal of Cell Science 108: 581-593. Author Disclosure Block: D. Chhabra, None; B. Hambly, None; N. Nosworthy, None; C.dos Remedios, None.

POS-L131 BOARD #LB131Length Scale Dependence of Actin Rheology and its Implication for Cell Mechanics.Jiayu Liu1, Margaret L. Gardel, PHD1, Klaus Kroy2, Erwin Frey3, Brenton D.Hoffman4, John C. Crocker4, David A. Weitz1. 1Harvard University, Cambridge, MA,USA, 2Hahn-Meitner-Institut, Berlin, Germany, 3Ludwig-Maximilians-UniversitätMünchen, Munich, Germany, 4University of Pennsylvania, Philadelphia, PA, USA.In entangled F-actin solutions, we vary the actin filament length by gelsolin, an actin sev-ering and cutting protein. By comparing one- and two-particle microrheology for sampleswith different filament length, we identify longitudinal density fluctuation modes as an

important mechanism of network elasticity over an extended frequency range of 0.01 to 30rad/sec. This suggests that one-particle microrheology may be useful for in vivo measure-ments of cells. This work also sheds light on how mechanics change during the sol-gel tran-sition. Author Disclosure Block: J. Liu, None; M.L. Gardel, None; K. Kroy, None; E. Frey,None; B.D. Hoffman, None; J.C. Crocker, None; D.A. Weitz, None.

POS-L132 BOARD #LB132Photo Patterning of Actin Filament Structures. Allen Liu, Daniel A. Fletcher, PhD.UC-Berkeley, Berkeley, CA, USA.We report a general strategy for spatiotemporal control of actin polymerization in vitrousing photoactivatable actin. Caged actin was synthesized by chemically modified lysineresidues on monomeric actin and released with focused ultraviolet (UV) illumination. Epi-fluorescence microscopy revealed nucleation and elongation of individual actin filaments (8nm in diameter) after localized release of caged actin. We also used this strategy to generatebranched filament structures by releasing caged actin in the presence of actin binding pro-teins. Controlled self assembly of actin filaments represents a versatile “bottom-up” tech-nique for constructing structural building blocks and functional templates for nanoscienceapplications. Author Disclosure Block: A. Liu, None; D.A. Fletcher, None.

POS-L133 BOARD #LB133The Nucleotide Switch in Cdc42 Modulates Coupling Between GTPase-Binding andthe Allosteric Equilibria of WASP. Daisy W. Leung, Michael K. Rosen. UTSouthwestern Medical Center, Dallas, TX, USA.The GTP/GDP nucleotide switch in Ras superfamily GTPases is generally considered aswitch of affinity towards downstream effectors, with the GTP-bound state having a high-er affinity for effector than the GDP-bound state. To better understand how GTPase bind-ing is coupled to effector activation, we have developed a quantitative model of allostericregulation of the Wiskott-Aldrich Syndrome Protein (WASP) by the Rho GTPase Cdc42.The model accurately predicts WASP affinity for Cdc42, activity towards Arp2/3 complex,and activation by Cdc42 as functions of a two-state allosteric equilibrium in WASP.Surprisingly, the ratio of GTPase affinities for the inactive and active states of WASP isappreciably larger for Cdc42-GTP than for Cdc42-GDP. This greater ability to distinguishbetween the two states of WASP enables Cdc42-GTP to behave as a full WASP agonist,while Cdc42-GDP is only a partial agonist. Thus, the nucleotide switch controls not onlythe affinity of Cdc42 for its effector, but also the efficiency of coupling between the Cdc42-binding and allosteric equilibria in WASP. This effect can ensure high fidelity and specifici-ty in Cdc42 signaling in crowded membrane environments. Author Disclosure Block: D.W.Leung, None; M.K. Rosen, None.

POS-L134 BOARD #LB134Probing the Role of the Actin Cross-linker Filamin A in Determining the MechanicalProperties of Living Cells. Karen E. Kasza, Nicholas A. Geisse, David A. Weitz.Harvard University, Cambridge, MA, USA.The actin binding protein filamin is implicated in many highly mechanical cellular process-es including cell locomotion, membrane blebbing, and mechanoprotection. Filamins con-stitute a large family of proteins that are abundant in many human tissues, with the FLNaisoform being the most widely expressed. Filamin cross-links actin filaments into orthogo-nal networks that are thought to provide structural stability to the cell. Because filaminbinds to a handful of macromolecules in the cell, including transmembrane proteins, it hasbeen proposed that filamin may be a key link between mechanical and chemical signalingpathways (Stossel et al, 2001). While some work has been done to understand how filaminaffects mechanical properties of in vitro reconstituted actin networks, little has been doneto probe its role in determining the mechanical properties of living cells. In order to eluci-date this mechanical role, we seek to compare the mechanical properties of a filamin-defi-cient human melanoma cell line (FLNa-) to a filamin-expressing subline (FLNa+) that hasbeen transfected with FLNa cDNA. We use the atomic force microscope (AFM) to per-form micro-indentation experiments on living FLNa- and FLNa+ cells. Using a simpleHertz-Sneddon model for elastic indentation to fit our AFM force-indentation data, wedetermine the indentation-depth dependent elastic moduli at many locations on each cell.We present a preliminary comparison of the measured mechanical properties of the FLNa+and FLNa- cell lines. Author Disclosure Block: K.E. Kasza, None; N.A. Geisse, None; D.A.Weitz, None.

POS-L135 BOARD #LB135Energetics and Dynamics of Actin Filament Bundling in a Network. Le Yang, DavidSept, Anders Carlsson. Washington University, St. Louis, MO, USA.The formation of filopodia-like bundles in vitro from a dendritic actin network has beenobserved to occur as a result of a nucleation process. We study both the energetics of theactin filament bundles and the dynamics of the actin filament ends in such a network inorder to evaluate the dynamics of the bundle nucleation process. Our model treats twosemiflexible actin filaments fixed at one end and free at the other, moving according toBrownian dynamics. The initial filament positions are chosen according to a thermal dis-tribution. We evaluate the dependence of the bundling time on the filament length andradius, and the distance between the filament bases. Since treating the movement of theindividual monomers in filaments is computationally unwieldy, we treat the filamentmotion using a normal mode analysis which permits use of a much longer timestep. Wefind that this method yields rapid convergence even when only the few longest-wavelengthmodes are included. Work supported by grants from the National Science FoundationDMS-0240770, the Whitaker Foundation and the National Institutes of Health RO1-GM067246. Author Disclosure Block: L. Yang, None; D. Sept, None; A. Carlsson, None.

POS-L136 BOARD #LB136PROXIMITY DETERMINATIONS BETWEEN SITES IN ACTIN-TROPOMYOSIN FILAMENT. Hui Wang, Shu Mao, Subhra Bhattacharya, GerardMarriott. UW-Madison, Madison, WI, USA.Previous FRET studies suggest that tropomyosin does not undergo significant movementsduring Ca2+-activation of reconstituted thin filaments (Tao et al., Biochemistry, 1983;

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Bacchiocchi et al., Biophys J., 2002). In those studies, wild-type tropomyosin (Tm) was ran-domly labeled with a donor probe at single cysteine residue (Cys-190) within a Tm dimer.The random labeling increases the uncertainty of FRET-based proximity determinationsbetween the donor probe on (Cys-190) and the acceptor probe on actin. In our study, wedetermined FRET efficiencies using a single donor probe labeled to a unique site on Tm -this was achieved using the homobifunctional crosslinking donor probe (bis-((N-iodoacetyl) piperazinyl) sulfonerhodamine) to covalently link the two free thiol groups inTm within wild-type Tm (Cys-190) and within several single cysteine mutants (TmS206C,TmT247C and TmT282C). SDS-PAGE analysis showed that a significant fraction of theseTm preparations contained crosslinked dimers as evidenced by the presence of a fluorescentband having the expected mass of 66 kD. The fluorescence emission arising from a popu-lation of non-crosslinked Tm having only a single donor probe was effectively quenched bysubsequent labeling with an excess of QSY-9-maleimide. G-actin was stoichiometricallylabeled with an acceptor probe (IC5-PE-maleimide) at Cys374, and polymerized to F-actinby adding an excess of phalloidin. FRET measurements between TMR at sires on on Tmand IC5 on actin showed that distances between the unique donor probe on Tm and theacceptor probe on actin were: 49.2 Å for Tm190C, 47.0 Å for TmS206C, 48.9 Å forTmT247C and 52.2 Å for TmT282C. This new approach improves the precision of FRETbased distance measurements from Tm and is being used in an ongoing study to map theCa2+-regulated movements of Tm and other proteins within reconstituted thin filaments.Author Disclosure Block: H. Wang, None; S. Mao, None; S. Bhattacharya, None; G.Marriott, None.

POS-L137 BOARD #LB137Human Platelet Profilin: Biophysical Charaterization Of A G-actin Binding Protein.Glendon D. McLachlan, PhD. Hunter College CUNY, Bronx, NY, USA.HPP is a G-actin binding protein that has been shown to co-localize with actin in the cellcytoplasm as well as the leading edge of motile cells. Profilin also binds polyphosphoinosi-tides and polyproline ligands in vitro. To better understand the structure/function paradigmin relation to HPP ligand interactions, characterization of the accessible conformationalstates of HPP by acid and urea titration has been performed.Results obtained by acid titration demonstrate that HPP unfolds via a three-state mecha-nism (N I U), with a highly populated intermediate between pH 4 and pH 5.Approximately 80% of native secondary structural content remains at pH 4, as determinedfrom monitoring the CD signal at 222 nm. There is a seven-fold increase in the fluorescencemaximum of the hydrophobic dye 8-Anilinonaphtalene-1-sulfonate (ANS) with a con-comitant blue shift in the maximum wavelength (?max), which indicates an exposedhydrophobic surface on the intermediate species. The stability (?GH2O) of the native con-formation at pH 7 and the intermediate species at pH 4, obtained from monitoring thechange in Trp and CD signal as a function of [urea], was determined to be 6.59 +/- 1.1 kcalmol-1 and 5.56 +/- 0.51 kcal mol-1 for the native state, and 4.01 +/- 0.62 kcal mol-1 and2.01 +/- 0.47 kcal mol-1 for the intermediate state. A residue specific description of the tran-sition from native to intermediate state was obtained by monitoring the equilibrium unfold-ing of HPP by 2D 1H-15N HSQC NMR spectroscopy. Stable intermediate species pop-ulate the NMR spectra between pH 4 and pH 5. A number of resonances show significantperturbations relative to the native spectrum, confirming that the pH 4 species represent anon-native equilibrium structure. Interestingly, the perturbed residues map to the G-actinand poly-L-proline binding surfaces on HPP.m Author Disclosure Block: G.D. McLachlan,None.

POS-L138 BOARD #LB138Structural Basis of Actin Filament Nucleation and Processive Capping by a ForminHomology 2 Domain. Takanori Otomo, Ph.D., Diana R. Tomchick, Ph.D., ChinatsuOtomo, Sanjay C. Panchal, Ph.D., Mischa Machius, Ph.D., Michael K. Rosen, Ph.D..University of Texas Southwestern Medical Center, Dallas, TX, USA.The conserved Formin Homology 2 (FH2) domain nucleates actin filaments and remainsbound to the growing filament barbed end. We have solved the crystal structure of theBni1p FH2 domain in complex with tetramethylrhodamine-actin. Each of the two struc-tural units (referred to as a bridge element) in the FH2 dimer binds two actins in an orien-tation similar to that in an actin filament, suggesting this structure could function as a fila-ment nucleus. Biochemical properties of heterodimeric FH2 mutants suggest the wild typeprotein equilibrates between two bound states at the barbed end, one permitting monomerbinding and the other permitting monomer dissociation. Interconversion between thesestates allows barbed end polymerization and depolymerization in the presence of boundFH2 domain. Kinetic and/or thermodynamic differences in the conformational and bind-ing equilibria can explain the variable activity of different FH2 domains, and the effects ofthe actin-binding protein profilin on FH2 function. Author Disclosure Block: T. Otomo,None; D.R. Tomchick, None; C. Otomo, None; S.C. Panchal, None; M. Machius, None;M.K. Rosen, None.

POS-L139 BOARD #LB139Biochemical and single molecule analyses of the dynactin:microtubule interaction.Stephen King, Tara Culver, Stephanie Lex. University of Missouri-Kansas City, KansasCity, MO, USA.Dynactin is an activator of dynein-based motility that can bind to microtubules plus ends,centrosomal microtubules near the microtubule minus ends, and along the length of micro-tubules during dynein motility events. We are examining the potential mechanism(s) bywhich dynactin can bind microtubules at specific cellular locations to have such diverse cel-lular functions. Using biochemical population assays, we found that the p150 subunit ofdynactin has two distinct microtubule-binding domains, a CAP-Gly domain and a basicdomain. In binding experiments, overexpression studies, and microtubule colocalizationstudies, the two domains have very similar properties. We have also performed single mol-ecule experiments with p150 fragments that contain each of the microtubule bindingdomains singly or in combination. In these experiments, the p150 fragments were attachedto polystyrene beads and the positions of protein-bound beads were followed as they boundto microtubules using particle-tracking software. We find that the CAP-Gly and basicdomains have distinct behaviors when they bind microtubules, which may reflect the poten-

tial roles of these domains in dynactin function. The observation that two microtubule-binding fragments with similar physiochemical properties in population assays have distinctproperties in single-molecule assays shows that our novel assay may be useful for examin-ing a variety of microtubule binding proteins. Author Disclosure Block: S. King, None; T.Culver, None; S. Lex, None.

POS-L140 BOARD #LB140Structural aspect of the interaction between dynein stalk and microtubule and its simi-larity to kinesin. Naoko Mizuno1,2, Masaki Edamatsu2, Kensuke Hirota2, Yoko Y.Toyoshima2, Masahide Kikkawa1. 1UTSW medical center, Dallas, TX, USA, 2Universityof Tokyo, Tokyo, Japan.Dyneins and kinesins move in opposite directions on microtubules. Because of the lack ofknowledge of the interaction between dynein and microtubules, not only the property ofdynein’s microtubule recognization mechanism, but also the question of how the sametrack is able to support bi-directional movement remains unanswered. To address this issue,we studied dynein-microtubule interactions using the tip of the microtubule-binding stalk,the dynein stalk head (DSH), which directly interacts with microtubules upon receivingconformational change from the ATPase domain. Biochemical and cryo-electron micro-scopic studies revealed that DSH bound to tubulin dimers with a periodicity of 80 Å, cor-responding to the step size of dyneins. The DSH molecule was observed as a globular corngrain-like shape that bound the same region as kinesin. Further three-dimensional recon-struction was done with the fragment that has longer coiled coil structure, DSH-SL(superlong) with microtubule. By comparing DSH and DSH-SL, we clearly saw the coiled coilportion of dynein stalk. Biochemical cross-linking experiments and image analyses of theDSH-kinesin head-microtubule complex revealed competition between DSH and thekinesin head for microtubule binding. The results demonstrate that dynein and kinesinshare an overlapping microtubule binding site, and imply that the binding at this site hasan essential role for these motor proteins.In this session, we will discuss two points, one is how dynein stalk, especially coiled coil por-tion, recognizes microtubule and the other is how microtubule can play a role as a rail ofdifferent molecular motors. Author Disclosure Block: N. Mizuno, None; M. Edamatsu,None; K. Hirota, None; Y.Y. Toyoshima, None; M. Kikkawa, None.

POS-L141 BOARD #LB141A Model of the Kinetic Cycle of Single Cytoplasmic Dynein Molecules. ManoranjanP. Singh, Ph.D, Roop Mallik, Ph.D, Steven P. Gross, Ph.D, Clare C. Yu, Ph.D. Univ. ofCalifornia, Irvine, Irvine, CA, USA.We use Monte Carlo simulations to model the molecular motor function of cytoplasmicdynein at the single molecule level. Our simulations reproduce the main features of recentsingle molecule experiments that found a discrete distribution of dynein step sizes depend-ing on load and ATP concentration. The theory relates dynein’s enzymatic properties to itsmechanical force production. It also requires the existence of a hierarchy of the ATP bind-ing affinities at the secondary binding sites, which is needed to reproduce the experimen-tally observed step size distribution and improve dynein’s ATP fuel economy by suppress-ing small steps under high ATP, no load conditions. On the other hand, in order to cap-ture the essential features of the step size distribution at very low ATP concentration and noload, the ATP hydrolysis of the primary site has to be dramatically reduced when none ofthe secondary sites have ATP bound to them. Author Disclosure Block: M.P. Singh, None;R. Mallik, None; S.P. Gross, None; C.C. Yu, None.

POS-L142 BOARD #LB142Cholesterol primes vascular smooth muscle to induce Ca2+ sensitization mediated by asphingosylphosphorylcholine/Src family tyrosine kinase/Rho-kinase pathway: possibleinvolvement of membrane lipid rafts. Hiroko Kishi, MD, PhD, Noriyasu Morikage,MD, Katsuko Kajiya, PhD, Hozumi Kawamichi, PhD, Sei Kobayashi, MD, PhD.Yamaguchi University School of Medicine, Ube, Japan.Hypercholesterolemia is a major risk factor of cardiovascular events. Rho-kinase (ROK)-mediated Ca2+ sensitization of vascular smooth muscle (VSM) plays a critical role in abnor-mal VSM contraction such as vasospasm. As an upstream mediator for such an abnormalpathway, we previously identified sphingosylphosphorylcholine (SPC), which induced theROK-mediated Ca2+ sensitization of VSM through the activation of Src family tyrosinekinase (Src-TKs). Therefore, in the present study, we examined the possible link betweenhypercholesterolemia (or cholesterol) and the Ca2+ sensitization of VSM mediated by aSPC/Src-TKs/ROK pathway. Tension study of VSM strips, which were obtained fromhuman patients and rabbit hypercholesterolemia model, showed that the extent of Ca2+

sensitization induced by SPC correlated well with total serum cholesterol concentration andwas diminished by either by cholesterol-lowering therapy for the patients or in vitro selec-tive depletion of cholesterol, but not of phospholipid, in VSM tissue by beta-cyclodextrin(beta-cdx). The immunofluorescent study using cultured VSM cells under confocal micro-scope showed that SPC induced the translocations of fyn, a member of Src-TKs, and ROKfrom cytosol to cell membrane, where they were colocalized with caveolin-1, a membraneprotein localized in cholesterol-rich membrane lipid rafts, or cholera toxin subunit B (CT-B), which selectively labels membrane lipid rafts. Beta-cdx treatment removed caveolin-1from cell membrane and markedly reduced the labeling of cell membrane by CT-B. Inthose beta-cdx-treated cells, the SPC-induced translocations of fyn and ROK from cytosolto cell membrane were diminished. These findings suggest that cholesterol and its enrichedmembrane microdomain, membrane lipid rafts, may be involved in Ca2+ sensitizationmediated by a SPC/Src-TKs/Rho-kinase pathway. Author Disclosure Block: H. Kishi,None; N. Morikage, None; K. Kajiya, None; H. Kawamichi, None; S. Kobayashi, None.

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POS-L143 BOARD #LB143AT THE ORIGIN OF THE RED EMISSION FORMS OF THE HIGHERPLANTS ANTENNA COMPLEX LHCA4. Roberta Croce1, Tomas Morosinotto2,Janne A. Ihalainen3, Milena Mozzo1, Agnieszka Chojnicka3, Frank van Mourik4, Jan P.Dekker3, Rienk van Grondelle3, Roberto Bassi5. 1CNR-IBF, Trento, Italy, 2Università diVerona, Verona, Italy, 3Vrije Universiteit, Amsterdam, The Netherlands, 4EPFL,Lausanne, Switzerland, 5Université Aix-Marseille II, Marseille, France. Photosystem I is characterized by red-shifted spectral forms. In higher plants most of theseforms are associated with the outer antenna which is composed by four Lhca complexes.Each of the Lhca1-4 subunit exhibits a specific fluorescence emission spectrum, peaking at688, 701, 725 and 733 nm respectively. Recent analysis revealed the role of chlorophyll-chlorophyll interactions of the red forms in all complexes. We report a detailed characteri-zation of the Lhca4 subunit using molecular biology, biochemistry and spectroscopy, andshow that the 733 nm emission form originates from a broad absorption band at 708 nm.Spectroscopy on recombinant mutant proteins assess that this band represents the low ener-gy form of an excitonic interaction involving two Chl a molecules bound to sites A5 andB5. These results are discussed on the basis of the structural information that recentlybecame available from X-ray crystallography (Ben-Shem et al. 2003). We suggest that, with-in the Lhca subfamily, spectroscopic properties of chromophores are modulated by thestrength of the excitonic coupling between the chromophores A5 and B5, thus yielding flu-orescence emission within a large wavelength interval. It is concluded that the interchro-mophore distance rather than the transition energy of the individual chromophores or theorientation of transition vectors represent(s) (the) critical factor(s) in determining the exci-tonic coupling in Lhca pigment-protein complexes. Author Disclosure Block: R. Croce,None; T. Morosinotto, None; J.A. Ihalainen, None; M. Mozzo, None; A. Chojnicka, None;F. van Mourik, None; J.P. Dekker, None; R. van Grondelle, None; R. Bassi, None.

POS-L144 BOARD #LB144Residual structure of the microtubule binding domain of the Alzheimer’s disease tauprotein. Patrick M. E. Barre, David Eliezer. Weill Cornell Medical College, New York,NY, USA.In the central nervous system, the tau protein exists as six isoforms. Each of these isoformscontain three or four imperfect repeat domains that interact with microtubules, favoringtheir polymerization and stabilization. In the Alzheimer’s disease brain however, tau is foundaggregated as straight and paired-helical filaments (PHFs) into neurofibrillary tangles.The structural NMR study presented here provides the first insight into the structure of tau,when bonded to microtubules or aggregated into filaments, at the level of individualresidues. Thus, our results show that the three-repeat domain of tau possesses α-helicalpropensities in regions corresponding closely to the sites of microtubule interactions.Besides, a stretch of residues previously associated with PHF initiation presents a propensi-ty for extended, β-sheet-like, structure. Furthermore, dimer formation through the estab-lishment of a disulfide bond is not required for the existence of these structural elements.By revealing the simultaneous presence of α-helical and β-sheet-like structures, these resultscan help to conciliate conflicting reports about the secondary structure of tau. AuthorDisclosure Block: P.M. Barre, None; D. Eliezer, None.

POS-L145 BOARD #LB145Characterization of proteins involved in multi component system mediated copper andsilver resistance. Ireena Bagai, Sylvia Franke, Megan M. McEvoy. University of Arizona, Tucson, AZ, USA.Both bacterial and eukaryotic cells contain an array of cytoplasmic membrane transport sys-tems involved in vital roles such as uptake of essential nutrients, excretion of toxic com-pounds and maintenance of cellular homeostasis.RND (resistance nodulation cell division) transport complexes are involved in export of awide range of organic compounds and heavy metal ions. Inside of the metal transportersthere are two groups of transporters. One transports divalent heavy metal ions (Co2+, Zn2+)and the other is involved in resistance against monovalent ions (Ag+, Cu+). The transportcomplexes consist of the cytoplasmic membrane RND protein, outer membrane factor(OMF) and membrane fusion protein (MFP). The best characterized RND silver/coppertransport complexes are Sil of Salmonella typhimurium pMG101 and Cus of E. coli. Uniquefor these transport complexes is the existence of an additional protein. In the Cus system,this additional protein was named CusF and shown to be located in the periplasm andinvolved in copper detoxification. Homologous systems identified in genome projects existseither as a discrete orf like CusF or in fusion with the MFP. This implies the likelihood ofan interaction of CusF with the MFP CusB. Interaction was analyzed using NMR andIsothermal Titration Calorimetry (ITC).The CusF homologue orf96aa of the silver resistance system Sil lacked a leader sequence forperiplasmic localization. However, if orf96aa (renamed SilF) were to accomplish the samefunction as CusF, a periplasmic localization is likely. The new sequence identified after rese-quencing of orf96 (accession number AF067954) did predict a leader sequence with thecleavage site identified using N-terminal sequencing. Initial characterization of SilF indi-cates silver binding and experiments directed at studying the differences in its affinity for sil-ver and copper binding have been performed using NMR. Author Disclosure Block: I.Bagai, None; S. Franke, None; M.M. McEvoy, None.

POS-L146 BOARD #LB146The Role of Cryoprotectants in Cryo-Cooling and Annealing of Protein Crystals.Douglas H. Juers1, Brian W. Matthews2. 1Whitman College, Walla Walla, WA, USA,2Howard Hughes Medical Institute, Eugene, OR, USA.Cooling of macromolecular crystals to cryogenic temperatures for X-ray diffraction datacollection often requires the presence of small molecules, or cryoprotectants, in the sup-porting solution. In some cases there is an optimal cryoprotectant concentration, at whichthe mosaicity and diffuse scatter are minimized and the diffraction limit is maximized. Weinvestigated the role of cryoprotectants in cryo-cooling by measuring the thermal contrac-tion of bulk solvents as a function of cryoprotectant concentration using a buoyancy basedtechnique. In general the thermal contraction of the bulk solvents increases with the cry-oprotectant concentration. Our analysis suggests that at the optimal cryoprotect concen-

tration, the contraction of the bulk solvent best compensates for the contraction of themacromolecular lattice. Too little or too much solvent contraction can both result in latticedamage. Additional studies suggest that in some cases, cycling crystals between low andhigh temperatures (annealing) can affect diffraction quality at low temperature by movingwater into or out of the crystal, which changes the cryoprotectant concentration and so thethermal contraction of the bulk solvent. Thus, the role of cryoprotectants appears not to beto prevent the formation of ice, per se, (as is commonly thought) but to tune the contrac-tion of the bulk solvent to match the inherent contraction of the macromolecular lattice.As the amount of solvent contraction depends on both the cryoprotectant and its concen-tration, these results point to the utility of screening both cryoprotectant identity and con-centration in the search for suitable cooling conditions. Author Disclosure Block: D.H.Juers, None; B.W. Matthews, None.

POS-L147 BOARD #LB147Analysis of Intermediate Resolution Structures. Matthew L. Baker, Ph.D., Wen Jiang,Ph.D., Wah Chiu, Ph.D. Baylor College of Medicine, Houston, TX, USA.Electron cryomicroscopy can now routinely produce subnanometer resolution reconstruc-tions for complex macromolecules. While electron cryomicroscopy has not yet reachedatomic resolution, a wealth of structural and functional information can be mined at thisresolution. To this end, we have developed the Analysis of Intermediate ResolutionStructures (AIRS) toolkit. AIRS incorporates several existing tools for structural analysis, aswell as new algorithms for feature extraction and analysis. Highlighting this toolkit isSSEHunter, a semi-automated method for simultaneous detection of alpha helices and betasheets. With the tools in AIRS, it is possible to construct pseudo-atomic structural modelsfor macromolecular structures at subnanometer resolutions. Additionally, AIRS provides aplatform for integrating ab initio and comparative modeling. AIRS is accessible through acommand line interface as well as a graphical interface in Chimera, which not only simpli-fies use but also provides additional functionality and online documentation.These tools have been successfully applied to Rice Dwarf Virus (RDV) and Herpes SimplexVirus-1 (HSV-1) major capsid protein, VP5. In Rice Dwarf Virus, both helices and sheetswere identified in the two structural proteins. In conjunction with sequence analysis andfold recognition, pseudoatomic models for these proteins were also determined and laterconfirmed by the x-ray structure. For HSV-1 VP5, the x-ray structure for only one of thedomains of the three domain major capsid protein was known. Again, using structural andsequence analysis, a pseudo-atomic model for the remainder of VP5 was determined, inwhich some structural similarity to lamboid bacteria phages structural proteins wasobserved in the floor domain of VP5. Additionally, ab initio modeling, constrained by thecryoEM density, allowed for the modeling of VP26, an accessory protein that binds onlyto hexonal VP5 molecules. Author Disclosure Block: M.L. Baker, None; W. Jiang, None; W.Chiu, None.

Pos-L148 Board #LB148Why Enzymes Work. Kevin Cahill, Ph.D. University of New Mexico, Albuquerque,NM, USA.A new hybrid potential shows why enzymes work. The hybrid potential is a combinationof the Rydberg form and the London inverse-sixth-power energy. It is more accurate thanthe harmonic and Lennard-Jones potentials and deeper at internuclear separations of 1-3Angstroms. Enzymes work because they need overcome only the lower energy barrier ofthe hybrid potential and not that of the harmonic or Lennard-Jones potential. AuthorDisclosure Block: K. Cahill, None.

POS-L149 BOARD #LB149Investigation of Enolase and Phosphoglycerate Mutase Interaction using MolecularDynamics Simulation. Davit Hakobyan, PhD student, Karen Nazaryan, PhD.Institute of Molecular Biology, Armenian National Academy of Sciences, Yerevan,Armenia.Glycolytic enzymes are responsible for conversion of glucose to pyruvate. It is well knownthat glycolytic enzymes can form dynamic complexes and substrate channeling in suchcomplexes may take place.CHARMM academic program has been used for energy minimization and leapfrog molec-ular dynamics to investigate further interaction between enolase and phosphoglyceratemutase (PGM) that has been shown experimentally in the in vitro conditions. To determinewhether this interaction is “visible” with the help of computer modeling method, two dif-ferent enolases have been chosen from available protein data banks. SaccharomycesCerevisiae PGM (1QHF) and enolase (2ONE) dimers have been tested for interaction.The results have been compared with results of interaction between Trypanosoma Brucei(TB) enolase monomer (1OEP) and the same yeast PGM. 3-phosphoglycerates of bothsubunits of PGM have been replaced with 2-phosphoglycerates (2PG). Enzymes wereplaced in a water box (with 10 Ao separation), which was additionally extended for 30 Aoin each X, Y and Z directions. Different orientations of enolase (30 degree step in each X,Y and Z direction) were used during simulation. For each orientation water box containedapproximately 25000 water molecules. During the simulation process, which took approx-imately 24192 CPU hours, the orientation of PGM was chosen to face its 2PGs to enolaseto put them in a favorable position for possible channeling. Comparison of results showedthat 66% of most active binding residues of TB enolase are identical to residues of yeastenolase. This is in a good agreement with Trypanosoma and yeast enolase residue sequenceidentity, which let us to assume this interaction to be species specific. Nevertheless no chan-neling has been recorded. Author Disclosure Block: D. Hakobyan, None; K. Nazaryan,None.

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POS-L150 BOARD #LB150Molecular Simulations of the Stability of An Alanine Octapeptide Confined in Small-Size AOT Reverse Micelles. ABEL Stephane, PhD student1,2, Waks Marcel1, UrbachWladimir3, Marchi Massimo4. 1Laboratoire d’Imagerie Parametrique, UMR 7623CNRS., Paris, France, 2Commissariat a l’Energie Atomique, DSV-SBFM-DBJC Centred’\’Etudes, Saclay, Gif-sur-Yvette, France, 3Laboratoire de Physique Statistique, CNRSUMR8550 Ecole Normale Superieure, Paris, France, 4Commissariat a l’ EnergieAtomique, DSV-SBFM-DJBC, Centre d’Etudes, Saclay, Gif-sur-Yvette, France.We have used atomistic Molecular Dynamics (MD) Simulations in NPT ensemble(T=300K and P=0.1MPa) to simulate a small sized Reverse Micelles (RM)(Wo=[H20]/[AOT]=5 and 7) with the ORAC MD package with/without a zwitterionicalpha-helix alanine octapeptide (known to adopt an unfolded conformation in water), con-fined in the micellar water core.We have examined the time evolution of the conformationalchange of the peptide in these systems compared to the same peptide in bulk water.We havecomputed the root-mean-square deviation from the initial structure and stability of intra H-bonds involved in alpha-helix structures (i.e CO(i)->NH(i+4) H-bonds) and analyzed theRM structures in presence/absence of the peptide (we have calculated the size of the watercore and of micelles with the gyration radius, the eccentricity, the radial mass density pro-files, the headgroups hydration, the area per surfactant, the micellar water diffusionetc.).The results indicate that the aggregate structural properties were significantely modi-fied for the small RM, in contrast to larger micelles.When the micelle is small and most ofthe available water is bound to AOT headgroups, the alpha-helical secondary structure ofthe peptide is conserved and stable in comparison with the same peptide in bulk water. Asthe quantity of water increases and the hydration water becomes available for the peptide,the formation of intermolecular H-bonds becomes possible, we observe then an unfoldingprocess.To test if the primary hydration of the polar headgroups of the surfactant can favorthe stability of peptide in the small micelle, we have removed the AOT headgroups. Thisresults in the loss of the peptide secondary structure.These simulations are in qualitativeagreement with experimental observations which indicate that hydration is a crucial param-eter for the stability of peptides/proteins confined in small-size AOT RM. Author DisclosureBlock: A. Stephane, None; W. Marcel, None; U. Wladimir, None; M. Massimo, None.

POS-L151 BOARD #LB151Ligand specificity of PR3 and HNE: a comparative molecular dynamics study. EricHajjar, Msc, Nathalie Reuter. University of Bergen, Bergen, Norway.The neutrophil serine proteinase 3 (PR3) is the specific target of anti-neutrophil cytoplasmantibodies (ANCA) in Wegener’s granulomatosis, a systemic necrotizing vasculitis. PR3shares high sequence homology (60%) with the human neutrophils elastase (HNE). Basedon these observation it has been stated that they have similar ligand specificities, they cleaveafter a small hydrophobic amino acid. While binding sites of PR3 have been described asmore restrictive than the corresponding ones in HNE (Fujinaga M., 1996) recent studiesshow that PR3 can cleave a broader range of substrates; peptides with a cysteine as the keyP1-site are specific to PR3 (Koehl C., 2003; Korkmaz B., 2003). Those recent studies alsosuggest an important role of the recognition sites situated after the cleavable peptide bond,i.e. the S’ sites, in contradiction with the idea that the enzyme specificity is controlled by theS1 and S subsites (Hedstrom L., 2002).Using molecular dynamics (MD) simulations, we investigated the substrate specificity ofPR3 and compared it to HNE. We modeled and simulated complexes between each of theenzymes and several peptides, known for being specific or not to PR3. An extensive analy-sis of the simulations: (i) show that the S1 pocket next to the catalytic triad of PR3 canaccommodate a cysteine; (ii) confirm the importance of the electrostatic interactionsbetween the S’ sites and the peptide; (iii) reveal an important flexibility of the S1 pocket andcooperativity between the binding subsites and S1.Using our results and the available experimental data we inventory the interactions betweenthe enzymes and the substrates. We finally propose a peptidic sequence pattern that wouldfit optimally and specifically to the PR3 binding sites. Author Disclosure Block: E. Hajjar,None; N. Reuter, None.

POS-L152 BOARD #LB152VAST Search: An improved online service for computing structural neighbors of pro-teins. Jie Chen, Ph.D., Tom Madej, Ph.D., Siqian He, Ph.D., Eric W. Sayers, Ph.D.,Stephen H. Bryant, Ph.D. National Center for Biotechnology Information, Bethesda,MD, USA.NCBI has released an improved web interface for VAST Search, a tool that allowsresearchers to submit a PDB file and retrieve structural neighbors to the query from EntrezStructure. VAST, an acronym for “Vector Alignment Search Tool,” rapidly scans candidatestructural alignments based on the relative orientations of axial vectors of secondary struc-ture elements, and then reports alignments exhibiting surprising similarity as judged by anobjective test statistic. To perform such a search, the new interface allows the user to entersimply the name of a PDB file. VAST Search will first convert the data to ASN.1 formatand then annotate 3D Domains on the protein fold. The 3D domains will be displayedgraphically on a validation page using the familiar MMDB format, along with a RequestID (RID), a unique integer that will serve as a tracking number for the VAST Search results.At this point either the converted structure may be viewed using Cn3D or saved to disk, ora VAST Search may be started. The status of the search can be viewed by entering the RIDon the main VAST Search page. When the search is complete, the results will be showngraphically using the same formatting as seen on the VAST Summary pages for pre-com-puted searches. The results page offers numerous options, including sorting the list of struc-tural neighbors, finding specific neighbors in the list, viewing the structural alignments withCn3D, and downloading the alignment in FASTA or ASN.1 formats. The latter formatalso includes the rotation matrices used to align the structural models in accordance withthe results of VAST, allowing the alignment to be reconstructed in other modeling envi-ronments. Author Disclosure Block: J. Chen, None; T. Madej, None; S. He, None; E.W.Sayers, None; S.H. Bryant, None.

POS-L153 BOARD #LB153Extracting chemically accurate small molecules from PDB. Paul A. Thiessen, Ph.D.,Eric W. Sayers, Ph.D., Stephen H. Bryant, Ph.D. National Center for BiotechnologyInformation, Bethesda, MD, USA.Described here are our efforts at bringing small molecules from PDB into the new NCBIPubChem database, using data from MMDB. The main challenge here is that PDB doesnot contain explicit bond orders, and often leaves out hydrogens. Even basic element iden-tity and atom connectivity are often suspect. While this is not much of a problem for pro-teins whose chemical structure is quite uniform, the unbounded variety of PDB’s hetero-gens makes filling in their chemical detail a more difficult task. We rely on OpenEye’sOEChem software library to do 3D coordinate-based perception, in order to obtain mol-ecules with complete chemical detail without manual intervention. Also discussed are howwe decide what constitutes a “small molecule,” a variety of problems encountered along thispath, and tricks used to overcome noise in the data. Author Disclosure Block: P.A. Thiessen,None; E.W. Sayers, None; S.H. Bryant, None.

POS-L154 BOARD #LB154Comparison of basis-set and method dependence of ground- and excited-state calcula-tions for cytosine and 5-methyl-cytosine in an excited-state quantum chemical investi-gation. Mark A. Jack, Ph.D. Florida A&M University, Tallahassee, FL, USA.A detailed computational study is performed on the molecular geometry and electronicspectra of cytosine and 5-methyl-cytosine. Ground-state geometries are optimized andcompared for each molecule for three different theoretical methods: Harttree-Fock theory(HF), density functional theory (DFT/B3YLP) and Moller-Plesset perturbation theory(MP2). For each method, the molecular ground-state energies and harmonic vibrationalfrequencies are calculated using four different basis sets, 6-31G(d,p), 6-31++G(d,p), 6-311G(d,p), and 6-311++G(d,p), in order to study the basis-set dependence of the calculat-ed observables. For the optimized ground-state geometries the vertical transition energies tothe excited states are computed using time-dependent density functional theory (TDDFT).These studies are conducted both for the gas phase and with water as a solvent in order tostudy the effects of hydration theoretically using different basis functions and methods.Effects of hydration on the geometries and spectra of the cytosine molecule are importantto study the structural stabilization and electronic excitations of DNA in water comparedto the gas phase. Such a study is especially crucial in the light of providing with TDDFT afast and precise computational alternative to the more accurate but computationally muchmore intensive coupled-cluster methods (CCSD(T)). All calculations on geometries, fre-quencies and spectra are done with the suites of the program Gaussian 03. The analysis ofthe molecular orbital transitions is done with the help of the molecular viewing programMolden, which also provides the initial molecule structures of cytosine and 5-methyl-cyto-sine as input for Gaussian. Author Disclosure Block: M.A. Jack, None.

POS-L155 BOARD #LB155Fragment Molecular Orbital method (FMO) and its application to biological mole-cules. Yuto Komeiji1, Dmitri Fedorov1, Tadashi Nemoto1, Kazuo Kitaura1, KaoriFukuzawa2, Shigenori Tanaka3, Tatsuya Nakano4. 1AIST, Tsukuba, Japan, 2Mizuho IRI,Tokyo, Japan, 3Kobe Univ, Kobe, Japan, 4NIHS, Tokyo, Japan.Computation of the electronic state of large molecules has recently become possible by theFragment Molecular Orbital (FMO) method [1]. FMO is a highly parallelizable and scal-able method, and is therefore suitable for large scale ab initio MO calculations of biologicalmolecules.The FMO method has been implemented into three MO programs: Gaussian 94 [1],ABINIT-MP [2], and GAMESS [3]. The ABINIT-MP program has been merged with amolecular modeling package PEACH to implement the FMO-MD method [4, 5].In this poster, recent advances in the FMO methodology are reviewed. Then, FMO com-putations of the pheromone binding protein and Catabolite Repressor Protein-DNA com-plex are presented. The applicability of FMO in biomolecular compuation and simulationwill be discussed.[1] Kitaura, K., et al., Chem. Phys. Lett. 313 (1999), 701.[2] Nakano, T., et al. Chem. Phys. Lett. 351 (2002), 475.[3] Fedorov, D. G., et al., J. Comput. Chem. 25 (2004), 873.[4] Komeiji, Y., et al., Chem. Phys. Lett. 372 (2003), 342.[5] Komeiji Y., et al., Comput. Biol. Chem. 2 (2004), 155.Author Disclosure Block: Y. Komeiji, NAREGI Research grants; D. Fedorov, NAREGIResearch grants; T. Nemoto, None; K. Kitaura, NAREGI Research grants; K. Fukuzawa,None; S. Tanaka, CREST Research grants; T. Nakano, FSIS Research grants; CREST Researchgrants.

POS-L156 BOARD #LB156Structural Model of the Rev Regulatory Protein from Equine Infectious Anemia Virus(EIAV). Yungok Ihm, Ph.D, Wendy Sparks, Jae-Hyung Lee, Yvonne Wannemuehler,Michael Terribilini, Haibo Cao, Cai-zhuang Wang, Susan Carpenter, Kai-Ming Ho,Drena Dobbs. Iowa State University, Ames, IA, USA.Rev is an essential regulatory protein in the equine infectious anemia virus (EIAV) and otherlentiviruses, including HIV. It binds incompletely spliced viral genomic RNA and exportsit from the nucleus to the cytoplasm, a critical prerequisite for the production of viral struc-tural proteins. Despite its important role in production of infectious virus, the developmentof antiviral therapies directed against Rev has been hampered by the lack of an experimen-tally-determined structure of the full length protein. We have used a combined computa-tional and biochemical approach to generate and evaluate a structural model of the Rev pro-tein. The EIAV Rev structure, modeled using a novel threading algorithm, includes a totalof 6 helices, four of which form an anti-parallel 4-helix bundle. The leucine-rich NES islocated in the first helix, with several hydrophobic side-chains directed outward, consistentwith their proposed interaction with the CRM1 export protein. An arginine-rich RRDRWmotif proposed to participate in RRE binding is located in a solvent-exposed loop. A sec-ond putative RNA binding motif, QERLE, is buried in the protein core, suggesting thatthe reduction in Rev function observed upon mutation of this motif may be due to desta-

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bilization of Rev, rather than direct abrogation of RNA binding. This interpretation is sup-ported by in vivo Rev activity assays and in vitro RNA binding experiments. The predictedRev structure may provide insights into the structural basis for common functions oflentiviral Rev proteins and suggest new antiviral strategies. Author Disclosure Block: Y. Ihm,None; W. Sparks, None; J. Lee, None; Y. Wannemuehler, None; M. Terribilini, None; H.Cao, None; C. Wang, None; S. Carpenter, None; K. Ho, None; D. Dobbs, None.

POS-L157 BOARD #LB157Improving Prediction of Transmembrane Segments of Integral Membrane ProteinsUsing Secondary Structure and Relative Solvent Accessibility Predictions. BaoqiangCao1, Rafal Adamczak2, Mark Jarrell1, Jaroslaw Meller2. 1University of Cincinnati,Cincinnati, OH, USA, 2Biomedical Informatics, Children’s Hospital ResearchFoundation, Cincinnati, OH, USA.Prediction of transmembrane (TM) domains has become an important tool in annotationof newly sequenced genomes [1]. Most of the existing prediction methods are based on var-ious hydropathy scales that indicate the presence of hydrophobic residues with a propensi-ty for membrane environment. Here, we propose an alternative approach that uses pre-dicted (from sequence) relative solvent accessibility (RSA) and secondary structure (SS) inorder to facilitate the prediction of TM segments. In that regard, the RSA predictions,which indicate residues unlikely to be exposed to aqueous environment (i.e. residues that

are eitherburied in thehydrophobiccore of aglobular pro-tein, or alter-n a t i v e l y ,“buried” in amembrane)turn out to beparticularly

useful. As shown in Talbe 1, accurate RSA and SS prediction may significantly increase theoverall accuracy of TM domains prediction, which was recently shown to be much lessaccurate than previously thought [4].Reference:Wallin E. and von Heijne G., Protein Sci. 1998, 7(4):1029-38.Jayasinghe, S., et al., Protein Sci. 2001, 10:455-8.Adamczak, R., et al., Proteins 2004, 56(4):753-67.Kernytsky, A. and Rost, B., Nucleic Acids Res. 2003, 31(13):3642-4.Author Disclosure Block: B. Cao, None; R. Adamczak, None; M. Jarrell, None; J. Meller,None.

POS-L158 BOARD #LB158Classification of Extended Proteins from Proteome Datasets by a PredictorSOSUIdumbbell. Nobuyuki Uchikoga1, Runcong Ke2, Masashi Sonoyama2, ShigekiMitaku2. 1Nagoya University, VBL. School of Engineering, Nagoya, Japan, 2NagoyaUniversity, Department of Applied Physics, School of Engineering, Nagoya, Japan.Genomes still include many genes of unknown functions as orphan genes, which have nohomologous sequences. Structure of proteins, then, is useful for adding information aboutfunctions of orphan sequences. Physicochemical characters of amino acid sequences involvein the mechanisms of protein folding. This approach could apply to prediction of the pro-tein structures, whose sequences have no homologous sequences. The mechanism of struc-tural changes could also be considered by such approach. We then have developed a methodof classification of protein structures by using physicochemical properties of amino acidresidues.Structure of extended proteins has two separate terminal domains linked by a central regionlike as calmodulin and troponin C. Amino acid sequences of extended proteins have largeexcess positively or negatively charged residues. Charged residues were distributed through-out in amino acid sequences. Therefore, long-range electrostatic interactions between twoterminal domains seem the dominant factor of mechanism of stabilizing extended struc-tures. Moreover, a central region linking between two terminal domains interacts with watermolecules and then amphiphilic residues are actually abundant in this region. These twointeractions in amino acid sequences could stabilize extended structure. These conditionscould classify extended structure from amino acid sequences deposited in a protein struc-ture dataset.Our method was applied to complete genome sequences. Predicted sequences seemed tohave extended structures and involved in regulatory functions. Half of predicted sequenceswere unknown proteins. Most of proteins with known functions were DNA-binding relat-ed proteins. Genomes of eukaryotes had more extended proteins than prokaryotes. Ineukaryotes, extended proteins were found in genomes of plants and vertebrates. Then, theevolution of genomes could be related with acquisition of extended proteins working as reg-ulating functions. Author Disclosure Block: N. Uchikoga, None; R. Ke, None; M. Sonoyama,None; S. Mitaku, None.

POS-L159 BOARD #LB159Fold recognition by combining sequence profiles derived from evolutionand from depth-dependent structural alignment of fragments. Hongyi Zhou, Ph.D,Yaoqi Zhou, Ph.D. SUNY at Buffalo, Buffalo, NY, USA.Recognizing structural similarity without significant sequence identity is proved to be achallenging task. Sequence-based and structure-based methods as well as their combina-tions have been developed. Here, we propose a fold recognition method that incorporatesstructural information without the need of sequence-to-structure threading. This is accom-plished by generating sequence profiles from protein structural fragments. The structure-derived sequence profiles allow a simple integration with evolution-derived sequence pro-files and secondary-structural information for an optimized alignment by efficient dynam-ic programming. The resulting method (called SP^3) is found to make a statistically sig-

nificant improvement in both sensitivity of fold recognition and accuracy of alignment overthe method based on evolution-derived sequence profiles alone (SP) and the method basedon evolution-derived sequence profile and secondary structure profile (SP^2). SP^3 wastested in SALIGN benchmark for alignment accuracy and Lindahl, PROSPECTOR 3.0,and LiveBench 8.0 benchmarks for remote-homology detection and model accuracy. SP^3is found to be the most sensitive and accurate single-method server in all benchmarks test-ed where the methods are available for comparison (although in some cases, its results arestatistically indistinguishable from the next best). In LiveBench 8.0, its accuracy rivals (or isbetter than) some of the consensus methods such as ShotGun-INBGU, Pmodeller3,Pcons4, and ROBETTA. Preliminary assessment of SP^3 server at CASP6 also shows itsoutstanding performance among all consensus and single-method servers. SP^3 fold recog-nition server is available on http://theory.med.buffalo.edu. Author Disclosure Block: H.Zhou, None; Y. Zhou, None.

POS-L160 BOARD #LB160Zipping: a fast conformational search strategy for protein folding. Vincent Voelz, KenDill. University of California-San Francisco, San Francisco, CA, USA.The Hydrophobic Zippers (HZ) model of protein folding proposes that fast folding can beachieved by making a series of local, low-entropy-loss contacts. In this way, proteins can findtheir global optimum (native state), not by performing a simultaneous optimization on allN degrees of freedom, but by doing a series of local optimizations instead, with a huge gainin search efficiency. Can this Folding Principle serve as specific recipe for ab initio folding?By examining simplified lattice models—where we can draw rigorous conclusions—we aimto determine how a limited search method based on zipping can find globally optimalstates. Preliminary evidence shows that often only small searches are required. Our ultimateaim is a method that can be used in all-atom searching to fold proteins via physical mod-els, and we are currently developing methods to examine protein contact space along zip-ping pathways using molecular simulation. Replica-exchange molecular dynamics simula-tions of Protein G (2GB1) and SH3 domain (1SRL) have been useful test cases in whichto examine zipping pathways and protein contact cooperativity. Author Disclosure Block: V.Voelz, None; K. Dill, None.

POS-L161 BOARD #LB161Prediction of Protein-Protein Interface Residues. Taner Z. Sen, PhD, AndrzejKloczkowski, PhD, Robert L. Jernigan, PhD, Changhui Yan, Vasant Honavar, PhD, Kai-Ming Ho, PhD, Cai-Zhuang Wang, PhD, Yungok Ihm, PhD, Haibo Cao, PhD, XunGu, PhD, Drena Dobbs, PhD. Iowa State University, Ames, IA, USA.The biological function of a protein is defined by its interactions with other molecules,including DNA, RNA, and small ligands, but especially with other proteins. These inter-actions are determined by the specificity and the structural proximity of amino acids locat-ed in the interface. The ability to predict these protein-protein binding sites would be a valu-able step in understanding protein function. However, the performance of different meth-ods for binding site identification can be limited depending on data availability, sequencespecificity, and three-dimensional structural constraints. In order to overcome the limitationof individual methods, we developed a two-rule consensus method based on four success-ful methods: conservatism of conservatism using homologous protein families, data min-ing with support vector machines (SVM), phylogenetic detection of conserved sites, andthreading through interfacial surfaces. We tested our consensus method on 7 protease-inhibitor pairs. Our results show that although certain individual methods can performwell, binding sites predicted by the consensus approach correlate better with experimentaldata. Author Disclosure Block: T.Z. Sen, None; A. Kloczkowski, None; R.L. Jernigan, None;C. Yan, None; V. Honavar, None; K. Ho, None; C. Wang, None; Y. Ihm, None; H. Cao,None; X. Gu, None; D. Dobbs, None.

POS-L162 BOARD #LB162Molecular Basis for Distinct Binding Modes of Different Drugs to the hERGPotassium Channel. Ashutosh A. Kulkarni, PhD, Mark W. Nowak, PhD, Nima Shiva,Niki M. Zacharias, PhD, John N. Nicholas, PhD, Heinte P. Lesso, Steven J. Reyes,Suedee Sahbha, Balji Lally, Elisha D. Mackey, Paul B. Bennett, PhD. NeurionPharmaceuticals, Pasadena, CA, USA.There is significant interest in understanding the molecular and biophysical determinantsof hERG potassium channel block. We have employed site-directed mutagenesis using nat-ural and unnatural amino acids to probe the interactions of therapeutically relevant drugswith the hERG pore (hERG MAPTM). We chose pore mutations that probe the steric,hydrophobic and electronic features that determine drug binding at amino acid positionsT623, S624, Y652, and F656. We also looked for backbone hydrogen bonding interactionsby mutating the peptide amide bond at L622 to an ester linkage. We expressed the mutat-ed channels in Xenopus oocytes and measured K+ currents by using two-electrode voltageclamp methods while perfusing the oocytes with various known hERG blockers. To evalu-ate the ability of the phenyl rings of Y652 and F656 to form cation-π interactions withdrugs, we incorporated into the channel unnatural amino acids which had mono-, di-, andtri-fluoro substitution on the rings. The substitution of electron-withdrawing fluorinesdecreases the cation-π binding ability of these residues. Risperidone and haloperidol showedcation-π interactions at Y652, whereas amperozide, fluphenazine, and triflupromazineshowed cation-π interactions at F656. Most of the drugs also exhibited hydrogen bondinginteractions with the backbone of L622 and the side chain of S624. We were able to clus-ter these compounds into distinct classes based on their binding interactions with the hERGchannel. We then made pharmacophore models for each cluster. Our data demonstrate thatdifferent molecules bind to the hERG channel in a variety of ways, and there is no univer-sal hERG binding pharmacophore. We are using this knowledge about hERG/drug inter-actions to synthetically eliminate the hERG binding ability of new therapeutic compounds.Author Disclosure Block: A.A. Kulkarni, None; M.W. Nowak, None; N. Shiva, None; N.M.Zacharias, None; J.N. Nicholas, None; H.P. Lesso, None; S.J. Reyes, None; S. Sahbha,None; B. Lally, None; E.D. Mackey, None; P.B. Bennett, None.

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POS-L163 BOARD #LB163Optical Detection and Tracking of Individual Gold Nano-Particles in Live Cells.Gerhard A. Blab, David Lasne, Stéphane Berciaud, Laurent Cognet, Brahim Lounis.Université Bordeaux 1, Talence CEDEX, France.Current state of the art imaging systems allow for the identification and the tracking of indi-vidual tagged molecules. Under carefully controlled conditions observation times of min-utes and positional accuracies down to 2 nanometers can be realized; for most applications,however, the rapid photo-bleaching (single fluorophores) or complicated photophysics(quantum dots) do not allow to take advantage of these limits.Using well established non-fluorescent markers in the form of nanometer-sized metal par-ticles, we present a novel method applicable for both assays and in vivo studies. PHI (pho-tothermal heterodyne imaging) is a simple, purely optical method utilizing the absorptiveproperties of metallic nano-particles close to their plasmon resonance. Gold aggregates assmall as 1.4 nm diameter (67 atoms), or even individual semiconductor quantum dots canbe detected by this method.We present data in which PHI is used to follow individual membrane proteins in vivo atvideo rate for several hours with minimal disturbance to the cell. The protein was labelledwith a 10 nm gold-bead attached via an antibody, resulting in a positional accuracy betterthan 20 nm. Such long trajectories allow not only to obtain diffusion constants but also forthe direct observation of changes between free diffusion and confinement in the same pro-tein over time. Author Disclosure Block: G.A. Blab, None; D. Lasne, None; S. Berciaud,None; L. Cognet, None; B. Lounis, None.

POS-L164 BOARD #LB164FLUCTUATION AND DISSIPATION IN A VISCOELASTIC MEDIUM. Olga S.Latinovic, M.S., H. Daniel Ou-Yang, Ph.D. Lehigh University, Bethlehem, PA, USA.It was proposed that one can determine the viscoealstic response function from thermalfluctuations. Despite attempts to validate the proposed approach, there has not been a directcomparison between the results obtained by the passive measurements and independentmicroscopic measurements which do not depend on thermal fluctuations. This paperreports a study that compares micromechanical responses obtained by both passive andactive measurements using the same probe particle. In the passive measurements, meansquare displacement and power spectral density of Brownian motions of a probe particle areused to obtain the viscoelastic modulus of poly(ethylene) oxide solutions using the general-ized Stokes-Einstein relation. In the direct, active measurements, the probe particle is setinto forced oscillations by oscillating optical tweezers. The viscoelastic moduli of the solu-tion are obtained from the displacements and the phase shifts of the forced oscillating par-ticle. We discuss the various ways to obtain the complex response function and compareresults obtained by these measurements. Author Disclosure Block: O. Latinovic, None; H.Ou-Yang, None.

POS-L165 BOARD #LB165Ultrastructural investigation of Mouse Sperm using Atomic force Microscopy andScanning Electron Microscopy. Sang-Joon Cho, Ph.D1, Sookyung Choi2, KipyoungJeon3, Myung Chan Gye, Ph.D.4.1Korea Advanced Institute of Science and Technology,Daejeon, Republic of Korea, 2PSIA Inc., Seoul, Republic of Korea, 3PSIA Inc.’, Seoul,Republic of Korea, 4Hanyang University, Seoul, Republic of Korea.The sperm cell is one of the simplest forms of eukaryotic cell containing only half of thewhole chromosome but it shows us its complex nature of a biological system. Nature even

designs sperm cells between species different-ly, probably by optimizing its design for opti-mal function for thousand years. The spermhead consists of acrosome containingdegradative enzyme and nucleus containing ahaploid set of chromosomes. The rest of thesperm parts are a middle piece containingmitochondria for energy and a tail for move-ment. By understanding the mechanism ofthis biological system, it could provide thepossible blueprint of nano- or micro-bio-mimetic robot for drug delivery. AFM is anideal tool to investigate the surface structureof sperm cell because a sperm head is flat andcan be attached on the slide glass firmly after

it is fixed or dried. We have studied the surface structure of mouse epididymal spermatozoaby using AFM and SEM. AFM and SEM pictures are compared and possible sample dam-ages caused by SEM are demonstrated. Author Disclosure Block: S. Cho, PSIA Inc.Consulting fees or other remuneration; S. Choi, PSIA Inc. Employment (full or part-time); K.Jeon, PSIA Inc. Employment (full or part-time); M. Gye, None.

POS-L166 BOARD #LB166Cell Motility as Persistent Random Motion: Theories from Experiments. DavidSelmeczi1, Stephan Mosler2, Peter Hagedorn2, Niels B. Larsen2, HenrikFlyvbjerg2.1Department of Biological Physics, Eötvös Loránd University, Budapest, Hungary, 2RisøNational Laboratory, Roskilde, Denmark.Experimental time series for trajectories of motile cells may contain so much informationthat a systematic analysis will yield cell-type-specific motility models. We show how here,using human keratinocytes and fibroblasts as examples. The two models that result, seemto reflect the cells’ different roles in the organism, and show that a cell has a memory of pastvelocities. Author Disclosure Block: D. Selmeczi, None; S. Mosler, None; P. Hagedorn, None;N.B. Larsen, None; H. Flyvbjerg, None.

POS-L167 BOARD #LB167Calorimetric Studies of Ligand Binding in R67 Dihydrofolate Reductase. MichaelJackson, M.S.1, Sally Chopra1, Derike Smiley1, Andre Rosowsky2, E.E. Howell1.1University of Tennessee, Knoxville, TN, USA, 2Dana-Farber Cancer Instistute, Boston,MA, USA.R67 dihydrofolate reductase (DHFR) catalyses the hydride transfer from nicotinamide ade-nine dinucleotide phosphate (NADPH) to dihydrofolate (DHF) reducing it to tetrahydro-folate, an important intermediate for DNA synthesis. R67 DHFR is a homotetramer pos-sessing 222 symmetry and has a single active site pore traversing the center of the toroidshaped molecule. R67 DHFR can bind a total of 2 ligands, either 2 folate/DHF moleculesor 2 NADPH molecules in binary, nonproductive complexes or 1 NADPH plus 1folate/DHF in a productive ternary complex. Isothermal titration calorimetry was used tomonitor the binding of cofactor and substrate analogs, inhibitors and fragments to refineand probe the important binding contacts with the enzyme. Ki studies were done to explorethe role of some analogs as inhibitors of R67 DHFR. In general, connectivity is importantfor both substrate and cofactor binding. While a gradual loss in binding affinity is observedas cofactor size is decreased, only minimal enthalpic signals are observed for folate fragmentssuch as pteroic acid and dihydrobiopterin that lack the glutamic acid tail. The substraterequires its charged glutamate tail for binding, but the further addition of 1-4 glutamatemolecules does not enhance binding.Interligand interactions may play a role in pairing preferences and result in observed differ-ences between homoligand (binary) and heteroligand (ternary) complex formation. Forexample, a binding comparison using four molecules, folate, N10-methyl folate,aminopterin, and methotrexate (MTX) together represent a thermodynamic cycle.However, binding studies do not show additive effects. Observations suggest a role for theO4 atom of folate in a pairing preference with NADPH. Ki studies also reflect this impor-tance for the O4 atom in folate analogs acting as inhibitors and show that folate analogswith changes in the pteridine’s B ring act as stronger inhibitors. Author Disclosure Block: M.Jackson, None; S. Chopra, None; D. Smiley, None; A. Rosowsky, None; E. Howell, None.

POS-L168 BOARD #LB168Development of hydrogel microfabrication techniques as experimental methods forconstructional approach to the morphological functions of multicellular systems.Hiroyuki Moriguchi, Akihiro Hattori, Tomoyuki Kaneko, Kenji Yasuda. University ofTokyo, Tokyo, Japan.Background & Purpose: Multicellular systems show both dependence and independenceon their morphological aspects, such as total shape, number or arrangement of a variety ofcells. To evaluate the effect of morphological aspects on a multicellular system, these mor-phological elements should all be considered and analyzed. From this point of view, wedeveloped morphological experimental techniques which enable controlling and perturb-ing the morphological aspects of living multicellular organisms, which use no-effectivehydrogels as the materials for holders of cultured living cell groups, and evaluated the valid-ity.Materials & Methods: We measured resolution, size range and laser-power dependency offour types of agarose-gel microfabrication techniques; 1) micromolding, 2) photo-thermaletching with focused 1064-nm laser beam, 3) photo-thermal etching with focused 1480-nm laser beam and 4) photo-thermal microneedle etching, by using 3-dimensional visual-ization with a conforcal microscope. Subsequently, we evaluated the ability of shape controlof living multicellular organisums with continuous observation of cells cultured in themicrostructures.Results: The resolutions of each technique were all higher than 2um. Cells in agarose-gelmicrostructures made no adhesion onto the surface of gel and kept their positions contin-uously. Each of the microfabrication techniques had advantages and limits; 1) micromold-ing is used to control 1- or 2-dimensional morphology of living multicellular organisms andhas advantage of high-throughput fabrication, 2) photo-thermal etching techniques are alsoused to control 1- or 2-dimensional morphology of living multicellular organisms and haveadvantages of the possibility of microfabrication during cell cultivation without clean-roomapparatus, and 3) photo-thermal microneedle etching is used to control 3-dimensionalshape of living multicellular organisms. Author Disclosure Block: H. Moriguchi, None; A.Hattori, None; T. Kaneko, None; K. Yasuda, None.

POS-L169 BOARD #LB169A Nanophysiometer for physiological measurements on single adult cardiac myocytesconfined in a sub-nanoliter volume on planar microelectrodes. Andreas A. Werdich,Eduardo A. Lima, Mark E. Anderson, Franz J. Baudenbacher. Vanderbilt University,Nashville, TN, USA.The Nanophysiometer is a versatile tool to measure multiple physiological parameters fromsingle, non-attached primary cardiac myocytes in a selectivly perfused microscopic cham-ber. We fabricated a microfluidic network with active valves in a silicone elastomer (PDMS)and combined it with planar microelectrodes deposited on a thin glass substrate. Thetranslucent microfluidic device was imaged with an inverted microscope and two glass fiberarays coupled to photomultipliers for high bandwidth optical recordings using multipleflourescencent dyes. Single cardiac myocytes were trapped inside the 250 pl microchamberby pressure gradients and maintained for several hours by continuous perfusion. Usingmicrofluidic channels we demonstrated the localized delivery of a fluorescent dye to a por-tion of a cardiac cell by measuring the resulting concentration gradient across the cell mem-brane. We also used the microelectrodes to generate well defined electric field gradients forstimulation and succeeded to pace single heart cells at a maximum frequency of 6.7 Hz andat physiological temperatures. We measured extracellular potentials as well as spatiallyresolved transmembrane potentials and calcium transcients to study calcium induced calci-um relase and sarcoplasmic reticulum Ca2+ load. Our device architechture can be expand-ed to allow complex perfusion protocols and simultaneous optical and electrochemical mul-tiparameter recordings of single cells. Author Disclosure Block: A.A. Werdich, None; E.A.Lima, None; M.E. Anderson, None; F.J. Baudenbacher, None.

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POS-L170 BOARD #LB170Metabolic flux analysis in mitochondria. Thuy D. Vo, Ines Thiele, Nathan D. Price,Bernhard O. Palsson. University of California, San Diego, La Jolla, CA, USA.Diverse datasets, including genomic, proteomic, isotopomer, and DNA sequence variation,are becoming available for human mitochondria. Thus there is a need to integrate these datawithin an in silico modeling framework, where mitochondrial biology and related disorderscan be studied and analyzed. We have reconstructed and characterized the human mito-chondrial metabolic network based on proteomic and biochemical data. The capabilities ofthe reconstructed network to fulfill three metabolic functions - oxidative phosphorylation,heme synthesis, and mixed phospholipid synthesis - were determined. Linear programmingand Monte Carlo sampling methods were applied to identify candidate steady states thatwere consistent with the imposed physiological and chemical observations. The activity ofthe mitochondrion was studied under four metabolic conditions: normal, diabetic,ischemic, and dietetic. Network-based analysis of the mitochondrial energy conversionprocess showed that the overall ATP yield per glucose is 31.5. Analyses of the steady-stateflux distributions showed that the experimentally observed reduced activity of pyruvatedehydrogenase in diabetic and ischemic patients could be a result of stoichiometric con-straints, and may not necessarily require enzymatic inhibition. Evaluation of the impact ofpreviously suggested treatments for diabetes showed that neither normalized glucose uptakenor decreased ketone body uptake have a positive effect on energy metabolism or networkflexibility. In summary, this study showed that an in silico framework for integrating multi-ple datasets is useful for studying mitochondrial metabolism under different stress condi-tions and provides a basis for assessing effects of potential disease treatments. AuthorDisclosure Block: T.D. Vo, None; I. Thiele, None; N.D. Price, None; B.O. Palsson, None.

POS-L171 BOARD #LB171Breaking the Patch Clamp Speed Barrier. Alan Finkel, Ph.D., James Costantin, Ph.D.,Andrew Wittel, Naibo Yang, Ph.D., Shawn Handran, Ph.D., Jan Hughes, Cathy Smith-Maxwell, Ph.D. Molecular Devices Corporation, Union City, CA, USA.Ion channels are a viable and under-represented target class in the pharmaceutical industry.Conventional patch clamping of a single cell at a time, the “gold standard” for studying ionchannels, is labor intensive and not amenable to screening large numbers of compounds inearly stage drug discovery. As a result, surrogate measures of ion channel activity, such as lig-and displacement, ionic flux and membrane potential are used for ion channel screening,but these assays have higher rates of false positives and negatives because one cannot con-trol the conformational state of channels due to the lack of voltage control. Recent advancesin planar patch clamp technology make it possible, for the first time, to automate electro-physiology for ion channel screening, and several instruments are now commercially avail-able. Although these systems offer considerably higher throughput than conventional patchclamping, there is still a pressing need for even higher throughput in order to drive downthe cost per compound tested and to match or exceed the throughput of the surrogateassays. An immediate four-fold improvement in throughput could be realized in theIonWorks HT system by eliminating the four-fold redundancy of the system, allowing over2000 data points to be collected per day. But to do so, the success rate would need toapproach 100% to be feasible. To achieve this goal, we developed the Population PatchClamp™ (PPC) technique, a revolutionary approach that records averaged ionic currentsfrom a population of cells expressing a recombinant voltage-gated ion channel. PPC exper-iments yielded extremely consistent recordings from well-to-well and success rates oftenexceeded 95% of the wells. I/V relationship and pharmacological characterization of Kv1.3and Nav1.5 channel data will be presented. Author Disclosure Block: A. Finkel, MolecularDevices Corporation Employment (full or part-time); J. Costantin, Molecular DevicesCorporation Employment (full or part-time); A. Wittel, Molecular Devices CorporationEmployment (full or part-time); N. Yang, Molecular Devices Corporation Employment (full orpart-time); S. Handran, Molecular Devices Corporation Employment (full or part-time); J.Hughes, Molecular Devices Corporation Employment (full or part-time); C. Smith-Maxwell,Molecular Devices Corporation Employment (full or part-time).

POS-L172 BOARD #LB172Optical tweezer and dielectrophoresis in studying the cell dielectric properties. EugeniaKovacs1, Dorel Pietreanu1, Mihai Radu1, Mersini Makropoulou2, AlexanderSerafetinides2, Eirini Papagiakoumou2.1Carol Davila Medical University, Bucharest,Romania, 2National Technical University, Athens, Greece.Dielectrophoresis and optical trapping are two methods for investigating and manipulatingmicroscopic particles and biological cells. If combined, these noninvasive techniques can beused to characterize the dielectric properties of individual living cells.A cell suspended in a medium with different dielectric properties becomes electrically polar-ized when subjected to an electric field. Interaction between the induced dipoles and thefield produces various electrokinetic effects, for instance dielectrophoresis; a spatially inho-mogeneous field will exert a lateral dielectrophoretic force on an uncharged particle.There is a relationship between dielectric properties and physiological state of the cell. Thus,a modification of cell functions can be determined via measurements of changes in dielec-trophoretic force being in equilibrum with an optical (tweezer) force, based on the assump-tion that optical force is invariant under modifications of dielectric parameters.A dielectrophoretic chamber with planar electrodes and an optical tweezer with a 830 nmlaser diode were used. Dielectrophoretic force equilibrating the optical force was measuredfor different cell lines(erythrocytes and yeasts). Analitical methods for determination ofdielectrophoretic force was based on Green’s theorem; numerical calculations were madeusing Mathematica 4.0 (Wolfram Research) software program. A cell was trapped in theoptical tweezer at a certain laser power. Increasing slowly the amplitude of the electric field,the dielectrophoretic force was thus increased until the cell was pulled out from the trap.This value of dielectrophoretic force approximates the optical force; the algorithm was usedfor control and modificated cells in order to provide a sensitive method to discriminatebetween normal and pathological cells using the modified values of Clausius Mossotti fac-tor. Author Disclosure Block: E. Kovacs, None; D. Pietreanu, None; M. Radu, None; M.Makropoulou, None; A. Serafetinides, None; E. Papagiakoumou, None.

POS-L173 BOARD #LB173Simultaneous fluorescence-interference detection for deconvoluting complex interac-tions at membrane. Martynas Gavutis, PhD student, Suman Lata, Peter Lamken, PiaMueller, Jan Dorr, Eva Jaks, Jacob Piehler. JW Goethe university, Frankfurt am Main,Germany.Signal propagation through the plasma membrane is mediated by complex cascades of pro-tein-protein interactions. These involve not only binding of ligands and effectors from solu-tion to transmembrane receptors, but also interactions within the plane of the membrane.Here, we describe an experimental approach for studying such interactions in a quantitativemanner. Simultaneous mass-sensitive and fluorescence detection was implemented by com-bining total internal reflection fluorescence spectroscopy (TIRFS) and reflectance interfer-ometry (RIf). Interferometric detection allows quantifying surface concentrations[mass/area] of interacting proteins, while fluorescence detection provides sensitive means formonitoring association and dissociation processes, as well as probing lateral interactions byfluorescence resonance energy transfer (FRET). An automated flow through system wasused allowing for resolving interaction rate constants up to 1-2s-1. The dynamics of ligand-induced assembling of the type I interferon receptor was investigated in detail using thisexperimental set-up. The extracellular domains the receptor subunits ifnar1 (ifnar1-EC) andifnar2 (ifnar2-EC) were tethered in an oriented fashion onto solid-supported, fluid lipidbilayers by using a novel, high-affinity chelator lipid. Thus, membrane anchoring and lat-eral diffusion of the receptor subunits was mimicked. Using ligand dissociation kinetics atdifferent receptor surface concentrations, the affinities of the ligand-receptor interaction inplane of the membrane was determined. Furthermore, the rate constants of these interac-tions were determined by using FRET. Strikingly, both association and dissociation rateconstants were highly dependent on membrane anchoring, corroborating the key role oforientation for efficient interaction at membranes. Author Disclosure Block: M. Gavutis,None; S. Lata, None; P. Lamken, None; P. Mueller, None; J. Dorr, None; E. Jaks, None; J.Piehler, None.

POS-L174 BOARD #LB174Silk Protein Polymer Block Design Rules. Cheryl T. S. Wong Po Foo, David L. Kaplan,PhD. Tufts University, Medford, MA, USA.Fibrous proteins offer an important system of study into how chemical information is uti-lized to control hierarchical structural assembly in Nature. For example, the study of theseproteins can open new doors toward the design of tailor-made materials with specific prop-erties, such as mimicking the impressive mechanical properties of silk fibers formed byinsects and spiders. However, what makes silk proteins so remarkable in design is the pre-dominance of hydrophobic amino acids to provide for hydrophobic interactions, waterexclusion, and beta-crystallite formation to produce the strong insoluble threads. Yet, theprotein domain structure of these protein designs also accommodate high solubility in aque-ous solution without premature precipitation as β-sheets during storage and processing.Recent studies have led to the hypothesis of a silk spinning mechanism starting with pro-tein chain folding to gel states before fibers are spun. This process has been mimicked invitro with regenerated silkworm fibroin along with varying concentrations of salts, polymerssuch as PEO, and control of water content. The detailed mechanisms controlling these fac-tors in the processing environment within the glands in vivo, in terms of how they affect theintra- and inter-chain interactions in silk fibroin chains, remain unclear. Therefore, a com-parative analysis of the distribution of charged amino acids in the N- and C-termini of silk,as well as the core repetitive region of silk proteins, was carried out to understand how theprocessing environment in vivo may influence protein folding. Furthermore, a series ofstructural variants of B. mori heavy and light chain fibroins was designed, cloned andexpressed to probe assembly behavior under different conditions to further elucidate theimportant factors controlling assembly. Author Disclosure Block: C.T.S. Wong Po Foo, None;D.L. Kaplan, None.

POS-L175 BOARD #LB175Single Molecule Detection in Optically Trapped Nanocontainers. Joseph E. Reiner,Alice M. Crawford, Rani Kishore, Kristian Helmerson. NIST, Gaithersburg, MD, USA.We demonstrate a new technique for the study of single molecules. We restrict the diffu-sion of single molecules to a submicron region by confining the molecule in a nanocon-tainer that can be held stationary by an optical trap. The confined molecule can then beinterrogated with high efficiency for an extended period of time. Optically trappednanocontainers offer many advantages over other single molecule techniques. The moleculein the nanocontainer is essentially free, but can be interrogated indefinitely unlike the situ-ation in free transit single molecule studies. In contrast with surface tethered molecules,molecules optically trapped in nanocontainers can be moved far from any surface that mayperturb the molecule of interest or contribute background fluorescence. The environmentin the nanocontainer can be quantitatively and rapidly changed by optically manipulatingtwo nanocontainers such that the contents mix.The nanocontainers we currently work with are surfactant stabilized, microscopic waterdroplets in a fluorocarbon medium, which we call hydrosomes. The solubility of water inthe fluorocarbon is sufficiently low that the hydrosomes are stable for several hours. The par-ticular fluorocarbon we are using has a lower index of refraction than water, which makesthe hydrosomes amenable to trapping by a single focused beam laser trap (optical tweezers).By using two independent optical traps, we can mix the contents of two hydrosomes bybringing them into contact, which causes them to fuse. Combining the optically trappedhydrosome with a laser excited, confocal detection set-up, we have been able to detect thefluorescence from single sulforhodamine B dye molecules. Evidence of the single moleculedetection is seen from the photobleaching step of the dye molecule held in the hydrosome.We are currently studying, via FRET, single molecule dynamics inside of a hydrosome.Author Disclosure Block: J.E. Reiner, None; A.M. Crawford, None; R. Kishore, None; K.Helmerson, None.

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POS-L176 BOARD #LB176Electromagnetic Force Instrument for Probing Site Specific Protein-DNA BindingKinetics by Unzipping DNA. Steven J. Koch, Ph.D.1, Gayle E. Thayer, Ph.D.1, JamesE. Martin, Ph.D.1, Bruce C. Bunker, Ph.D.1, James H. Werner, Ph.D.2, Peter M.Goodwin, Ph.D.2, Richard A. Keller, Ph.D.2, George D. Bachand, Ph.D.1. 1SandiaNational Laboratories, Albuquerque, NM, USA, 2Los Alamos National Laboratory, LosAlamos, NM, USA.Forced disruption of a single molecular bond is a force-assisted, thermodynamically drivenevent—meaning that hundreds of single measurements must be acquired in order to extractinformation about the underlying energy landscape(1). While optical tweezers offer manyadvantageous features, including exquisite feedback control and pN/nm precision over along range of extensions, the assays operate on one molecule at a time and can be tediousfor obtaining statistically sufficient data. Magnetic field gradients from permanent magnetscan be used to apply constant forces to unzip many single DNA molecules simultaneous-ly(2), but mechanical translation of the magnets is required for force modulation. We areconstructing a computer-controlled electromagnetic instrument able to exert dynamicforces greater than 50 pN simultaneously on dozens of single molecules in order to unzipDNA molecules in the presence of DNA-binding proteins(3). We plan to use the instru-ment to investigate the effects of water on site-specific DNA binding kinetics of EcoRI andcompare these single molecule measurements with bulk methods(4). We will report pre-liminary measurements along with details of the instrument design and capabilities.Sandia is a multiprogram laboratory operated by Sandia Corporation, a Lockheed MartinCompany, for the United States Department of Energy’s National Nuclear SecurityAdministration under Contract DE-AC04-94AL85000. 1. Evans, E. (2001) Annu Rev Biophys Biomol Struct 30, 105-28.2. Danilowicz, C., Coljee, V. W., Bouzigues, C., Lubensky, D. K., Nelson, D. R. & Prentiss,M. (2003) Proc Natl Acad Sci U S A 100, 1694-9.3. Koch, S. J., Shundrovsky, A., Jantzen, B. C. & Wang, M. D. (2002) Biophys J 83, 1098-105.4. Sidorova, N. Y. & Rau, D. C. (2001) J Mol Biol 310, 801-16.Author Disclosure Block: S.J. Koch, None; G.E. Thayer, None; J.E. Martin, None; B.C.Bunker, None; J.H. Werner, None; P.M. Goodwin, None; R.A. Keller, None; G.D.Bachand, None.

POS-L177 BOARD #LB177MatLab Package with Graphical User-Interface for Efficient and Precise Calibration ofOptical Tweezers. Poul Martin Hansen1, Iva Marija Tolic-Norrelykke2, HenrikFlyvbjerg3, Kirstine Berg-Sorensen, Ph.D.1. 1University of Copenhagen, Copenhagen,Denmark, 2Max Planck Institute for Cell Biology and Genetics, Dresden, Germany,3Risø National Laboratory, Roskilde, Denmark.We present a MatLab (MathWorks Inc.) program with graphical user interface that cali-brates an optical trap. This is done by analysing timeseries of the position of a trappedmicrosphere, as described in Rev. Sci. Ins. 75, pp. 594-612 (2004). The program performsa number of health checks on the experimental data. Then the power spectrum is calculat-ed and the theory of choice is fitted to it. The theory optionally accounts for the frequencydependence of the hydrodynamic drag force, for hydrodynamic interaction with a nearbycover slip, for effects of finite sampling frequency (aliasing), for effects of anti-aliasing filtersin the data acquisition electronics, and for parasitic filtering caused by the position detec-tion system. Each of these effects can be left out or included as the user prefers. User-definedparameters may be used. The results are presented graphically, and values for fitted param-eters are given with statistical errors and covariance matrix. Bring your datafiles to the posterand we’ll help you analyse them with a few clicks on a mouse. Author Disclosure Block: P.Hansen, None; I. Tolic-Norrelykke, None; H. Flyvbjerg, None; K. Berg-Sorensen, None.

POS-L178 BOARD #LB178Stepwise bending of DNA by a single TATA-box Binding Protein. Simon F. Tolic-Nørrelykke1,2, Mette B. Rasmussen3, Kirstine Berg-Sørensen3, Lene B. Oddershede3.1Max Planck Institute, Dresden, Germany, 2Laboratory for Non-Linear Spectroscopy,Florence, Italy, 3Niels Bohr Institute, Copenhagen, Denmark.The TATA-binding protein (TBP) is required by all three eukaryotic RNA polymerases forthe initiation of transcription from most promoters. TBP recognizes and binds to TATAsequences in the DNA. The binding is in the minor groove in the DNA and the TBP-DNA interaction is considered a defining example of indirect readout. Upon binding, TBPpartially unwinds the DNA and bends it by ~80°. This distortion of the DNA is thoughtto play a crucial role in the recruitment and stabilization of the RNA polymerases. We pres-ent results from the study of individual TBPs interacting with single DNA molecules con-taining a TATA-box. Using video microscopy we observe the Brownian motion of a beadattached to a short surface-bound DNA tether. When the DNA tether is bound by TBPthe effective end-to-end distance is reduced and gives rise to a diminished amplitude ofBrownian motion. By monitoring the Brownian motion as a function of time we were ableto detect individual binding and unbinding events and measure rate constants for theprocess. Unbinding was induced by introducing a large amount of electrolyte or by direct-ly pulling on the tethered bead using optical tweezers. In addition to the well defined boundand unbound states we also observed a third state, intermediate in both time and spacebetween bound and unbound. These observations support a recently published model forthe reaction pathway in which an intermediate state is populated a large fraction of the time.Author Disclosure Block: S.F. Tolic-Nørrelykke, None; M.B. Rasmussen, None; K. Berg-Sørensen, None; L.B. Oddershede, None.

POS-L179 BOARD #LB179Real-time single flavoenzyme dynamics studied with fluorescence correlation spec-troscopy. Adrie H. Westphal, Andrey Matorin, Jan Willem Borst, Willem J. H. vanBerkel, Antonie J. W. Visser. Laboratory of Biochemistry, Wageningen University,Wageningen, The Netherlands.Flavoenzymes in general are not the easiest systems for single-molecule fluorescence studies.In most flavoenzymes the flavin has low molecular brightness due to severe quenching of

its fluorescence. To overcome this limitation a second, strongly fluorescent donor label canbe used that can communicate with the flavin via single-pair Förster resonance energy trans-fer (spFRET). The donor fluorescence properties can be used as a sensitive antenna forredox state and enzymatic activity of the flavoenzyme. The label can be designed such thatspFRET to the flavin will only affect the donor fluorescence when the flavin is in the oxi-dized state, whereas in the reduced state there is no longer spectral overlap. Single-moleculefluorescence detection can thus provide a detailed picture of the conformational dynamicsof single flavoenzymes and its relation to catalysis. We illustrate this approach using theflavoprotein p-hydroxybenzoate hydroxylase (PHBH) that carries out the hydroxylation ofthe aromatic substrate p-hydroxybenzoate (PHB). PHBH contains one accessible cysteinegroup per monomer. We have labeled Cys116 of PHBH with two different fluorescentmaleimides. One of these labels shows overlap between its fluorescence spectrum and theabsorption spectrum of the flavin prosthetic group, while the other fluorescent probe doesnot have this spectral overlap and serves as a control. The experiments consist of single-mol-ecule fluorescence correlation spectroscopy of the labeled enzymes in the absence and pres-ence of enzymatic turnover. Distinct changes in the time-dependent autocorrelation curvesof spFRET-active PHBH can be observed when this enzyme is performing catalysis usingboth substrates PHB and NADPH. The nature of this extra relaxation process is explored.Author Disclosure Block: A.H. Westphal, None; A. Matorin, None; J. Borst, None; W.J.H.van Berkel, None; A.J.W. Visser, None.

POS-L180 BOARD #LB180High Performance Magnetic Tweezers for Study of Single Macromolecules. Seok-Cheol Hong, Ph.D.1,2, David E. Humphries3,4, Michael D. Stone, Ph.D.5, Carlos J.Bustamante, Ph.D.2,6, Nicholas R. Cozzarelli, Ph.D.2. 1Korea University, Seoul, Republic of Korea, 2University of California, Berkeley, CA,USA, 3Engineering Division, Lawrence Berkeley National Laboratory, CA, USA, 4JointGenome Institute, Walnut Creek, CA, USA, 5University of California Berkeley, Berkeley,CA, USA, 6Physical Bioscience Division, Lawrence Berkeley National Laboratory,Berkeley, CA, USA.We report the development of magnetic tweezers capable of applying forces over a five-order-of-magnitude range from 10 fN to approximately 1 nN. This high force range wasachieved using hybrid supermagnets. Force measurements on DNA and glass micropipettesare in good agreement with theoretical estimations. The ~ 7 fold increase in force allows theuse of smaller magnetic beads with ~ 2 to 3 fold better time resolution. Additionally, weimplemented a fast analysis scheme involving Hilbert transformation that permits at least20 Hz real-time position measurement of DNA extension. These new features make mag-netic tweezers competitive with other manipulation tools in the high force regime whilemaintaining its superiority in the low force range and in applying torque. Author DisclosureBlock: S. Hong, Lawrence Berkeley National Laboratory; D.E. Humphries, Lawrence BerkeleyNational Laboratory; M.D. Stone, None; C.J. Bustamante, None; N.R. Cozzarelli, LawrenceBerkeley National Laboratory.

POS-L181 BOARD #LB181Probing Gene Expression in Single Live Sacchromyce cerevisiae Cells: One Molecule ata Time. Jialu Yin, Ji Yu, Jie Xiao, X. Sunney Xie. Harvard University, Cambridge, MA,USA.Quantitative understandings of genetic networks require the ability to probe protein con-centrations in real-time in vivo. Existing methodologies lack the sensitivity and time resolu-tion to probe single proteins in single live cells. We devised a protein reporter system thatenables explicit imaging of gene expressions in single live Saccharomyce cerevisiae cells withsingle protein sensitivity and time resolution of a few minute. The approach is based onmodified β-galactosidase enzymatic reporter protein with short cellular lifetime, whichhydrolyze a fluorogenic membrane-permeable substrate and gets degraded by proteasome.We have tested this reporter system with GAL1-10 promoter and hexose transporter(HXT) promoters under repressed culturing condition, and observed time-resolved fluo-rescently amplified signals. We recorded individual fluorescent bursts on individual cells thatresulted from transient gene expression. Modified β-galactosidase reporter system hasdemonstrated single protein sensitivity in single live cells, providing new kinetic informa-tion for quantitative studies of eukaryotic genetic regulation. We are applying this method-ology to study the expression profile of a family of HXT proteins, a model subset of S. cere-visiae genome, the systems-wise study of which will become exemplary in quantitative pro-filing of eukaryotic genetic regulation. Author Disclosure Block: J. Yin, None; J. Yu, None;J. Xiao, None; X.S. Xie, None.

POS-L182 BOARD #LB182Calibration of optical tweezers using a piezo-electric translation stage. Simon F. Tolic-Norrelykke1,2, Francesco S. Pavone2, Henrik K. Flyvbjerg3. 1Max Planck Institute,Dresden, Germany, 2LENS, Florence, Italy, 3Risoe National Laboratory, Roskilde,Denmark.We introduce a method to calibrate the position detection system, in the x-y plane, for opti-cal tweezers. The method relies on the use of a type of piezo-electric stage that is common-ly found in optical tweezers setups. By harmonically driving the piezo stage a known motionis added to a trapped object’s Brownian motion. This added motion serves as a “scale-bar”and thus enables the calibration of the detection system. It is not necessary to know frictioncoefficients, hence neither the viscosity or temperature of the surrounding liquid. Nor is anyknowledge required about the trapped object’s size, refractive index, or distance to nearbysurfaces. Author Disclosure Block: S.F. Tolic-Norrelykke, None; F.S. Pavone, None; H.K.Flyvbjerg, None.

POS-L183 BOARD #LB183Possible role of multi-states of proteins in robust and flexible functioning of signaltransduction system. Hiroaki Takagi. Osaka university, Suita, Japan.Relaxation of proteins is often quite slow process. From theoretical analysis, it was verifiedthat the relaxation time of proteins can be longer than the waiting time of successive inter-actions between proteins inside of cells (B.Hess and A.Mikhailov, 1994). It was also report-ed that some proteins have several meta-stable states and make transitions between them.

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Both points to be considered, here we discuss a possible role of multi-states of proteins incellular signal transduction: whether fluctuations of states in signaling proteins can ensureflexible and robust response to environmental changes or not.To pursue such a possibility, we extended slow conformational-relaxation model of a singleenzyme kinetics (H-P.Lerch et.al., 2002) to the one of a signaling molecule with multi-states, and investigate its behaviors numerically. Through the simulations, we found that thestate of signaling protein can be selected flexibly with amplification through interactionsbetween different effectors according to their concentration changes. There was some fitteststrength of fluctuations of states in signaling proteins that can realize such flexible behav-iors. Moreover, this results was rather robust against fluctuations of effector concentrations.Finally we discuss the possible relationship between the results and Ras signaling system.Author Disclosure Block: H. Takagi, None.

POS-L184 BOARD #LB184Development of plastic microfluidic mixer for protein folding studies. Hye Yoon Park,Lisa Kwok, Lois Pollack. Cornell University, Ithaca, NY, USA.We report a novel microfluidic diffusive mixer designed to investigate the rapid folding ofproteins and RNAs. Within this device, mixing is initiated uniformly and is complete onthe order of 100 microseconds. The characteristic time is determined by measuring the col-lisional quenching of fluorescein dye by iodide ions. Iodide ions have a relatively low diffu-sion coefficient; mixing will be much faster for other condition changes, such as a pH jump.Numerical and analytical solutions of diffusion within the device will be presented and areconsistent with the experimental data. The mixer is fabricated by imprinting on a transpar-ent plastic material. This practical method allows application of optical grade plastic mate-rial, low fabrication cost and short turnaround time. We anticipate that the mixer will be aconvenient tool in subsequent measurements of rapid conformational changes of fluores-cently labeled proteins using confocal and multiphoton microscopy. This work was sup-ported by the Nanobiotechnology center (NBTC), an STC program of the NationalScience Foundation. Author Disclosure Block: H. Park, None; L. Kwok, None; L. Pollack,None.

POS-L185 BOARD #LB185A family of site selective molecular optical switches. Tomoyo Sakata1, Yuling Yan2,Gerard Marriott1. 1University of Wisconsin-Madison, Madison, WI, USA, 2University ofHawai`i, Honolulu, HI, USA.We propose a new approach for optical switching of biomolecular interactions that is basedon site selectively labeling of photochromic probes to biomolecules. Spirobenzopyran andspironapthoxazine undergo efficient, rapid and reversible, optically-driven transitionsbetween a colorless spiro (SP) state and a brightly colored merocyanine (MC) state withoutthe release of photoproducts. In this presentation, we describe the design, synthesis andcharacterization of a family of thiol reactive spirobenzopyrans and spironapthoxazine withthe results of spectroscopic analysis of the conjugates with actin and BSA. Five thiol reac-tive spirobenzopyrans and one thiol reactive spironaphthoxazine were prepared by couplingindoline derivatives with the corresponding salycilaldehydes or 1-nitroso-2-naphthol. Thelocation or length of the thiol reactive group on a common chromophore differs for eachprobe. Spectroscopic analyses of 6 optical switch conjugates of actin and BSA were used tocharacterize the interactions between the protein and the probe in both MC and SP states.The highly polar MC state was shown to engage in specific dipolar interactions with theprotein, the strength of which depended on the MC linkage geometry. Dipolar interactionswithin the corresponding SP conjugates were far weaker and scaled with the value of thedipole moment. We demonstrate that optical switching between the SP and MC stateswithin these protein conjugates proceeds with high fidelity and the recovery of specific dipo-lar interactions. The MC absorption of a spironaphthoxazine conjugate is red shifted bymore than 100 nm compared to the equivalent spirobenzopyran, which may be exploitedfor the independent control of the MC to SP transition for upto two different spironaph-thoxazine and spirobenzopyrans conjugates within the same sample. Author DisclosureBlock: T. Sakata, None; Y. Yan, None; G. Marriott, None.

POS-L186 BOARD #LB186Human Embryonic Stem Cell-derived Cardiomyocytes Can be Maintained in Serum-Free Medium. Jia-Qiang He, Ph.D.1, Chunhui Xu, Ph.D.2, Joseph D. Gold, Ph.D.2,Shailaja Police, Ph.D.2, Xiaoming Hao, Ph.D.2, Melissa K. Carpenter, Ph.D.2, JaneLebkowski, Ph.D.2, Timothy J. Kamp, Ph.D. and M.D.1. 1University of Wisconsin-Madison, Madison, WI, USA, 2Geron Corporation, Menlo Park, CA, USA.Human embryonic stem (hES) cells can differentiate into functional cardiomyocytes (CM),which may be useful for cell transplantation, pharmacological testing, and multiple researchapplications. To understand and optimize in vitro cardiac differentiation in serum-free con-ditions, we investigated whether a serum-free medium containing creatine, carnitine andtaurine (CCT) can maintain hES cell-derived CM in comparison to standard serum medi-um. The expressions of cardiac specific genes and proteins were compared using TaqMananalysis and immunocytochemistry. The functional phenotypes of the cells were evaluatedby intracellular electrophysiology techniques and by measuring beating rate. TaqMan analy-sis showed a comparable expression of cardiac α-myosin heavy chain and Nkx2.5 from cellscultured in serum compared to CCT at 19 days of differentiation. Immunocytochemistryshowed the expression of cardiac troponin I, sarcomeric myosin heavy chain, N-cadherinand connexin43 in cells treated with CCT. In addition, beating cells in CCT responded toditiazem, isoprenaline and phenylephrine in a dose dependent manner. Furthermore, elec-trophysiological measurements in beating embryonic bodies (EBs) at days 19-22 of differ-entiation showed that the vast majority of cells display ventricular-like action potentials(APs), and the ventricular cells had indistinguishable AP properties comparing the twogroups: APD50/APD90 (AP duration measured at 50% or 90% repolarization) are388±72/506±57ms (Serum) vs 422±77/551±111ms (CCT); dV/dtmax (maximum rise rateof AP) are 10.3±3.8V/s (Serum) vs 9.2±1.7V/s (CCT); and MDP (maximum diastolicpotential) are -49.3±6.4mV (Serum) vs -48.1±5.6mV (CCT) (n=33 from 9 CCT-treated

EBs and n=62 from 12 Serum-treated EBs, p>0.05 between serum and CCT treatment forall AP parameters). These results show that hES cell-derived CMs maintained in CCTcompared to serum exhibit similar phenotypes. The culture of hES cell-derived CM inserum-free medium should facilitate the use of the cells for in vitro and in vivo applications.Author Disclosure Block: J. He, Geron Corporation Research grants; C. Xu, GeronCorporation Employment (full or part-time); J.D. Gold, Geron Corporation Employment (fullor part-time); S. Police, Geron Corporation Employment (full or part-time); X. Hao, GeronCorporation Employment (full or part-time); M.K. Carpenter, Geron CorporationEmployment (full or part-time); J. Lebkowski, Geron Corporation Employment (full or part-time); T.J. Kamp, Geron Corporation Research grants.

POS-L187 BOARD #LB187FTIR SPECTROSCOPIC APPROACH FOR PLANT TAXONOMY. SevgiGorgulu, MSc, Musa DOGAN, Feride SEVERCAN. Middle East Technical Uni.,Ankara, Turkey.Several techniques have been used to identify and classify plants based on morphological,anatomical, genetical and chemical characteristics. But there are still disagreements fordelimination of plant groups. New techniques which give information about molecularstructure of the system may bring fundamental evidence to improve systematic of plantsbecause chemical structures vary widely from a plant group to another.Fourier Transform Infrared Spectroscopy (FTIR) is proposed in this study as a powerfulltechnique for characterizing and identifying structure of cellular components to differenti-ate the genera namely Ranunculus, Acantholimon and Astragalus according to their molecu-lar structure by sampling the leaves of the plants.Ranunculus, Acantholimon and Astragalus are angiosperm plants whose some species growin Turkey. Ranunculus and Acantholimon have ornamental importance while Astragalus isan improtant pharmaceutical genus.FTIR study revealed dramatic differences in between these three genera which suggest thatAcantholimon has the highest hydrogen capacity in its polysaccarides and proteins, changesin lipid metabolism of each genus. An increase in lignin content and a decrease in pectinesterification and in decarboxylation processes for Ranunculus and Astragalus compared toAcantholimon have been observed. Moreover, different cell wall polysaccharides includingdiverse groups could be identified for each genus by this technique. This study presented anew method giving information at molecular level for rapid identificaiton in taxonomicstudies at the level of genus. Author Disclosure Block: S. Gorgulu, None; M. Dogan, None;F. Severcan, None.

Abstract Author DisclosuresCarpenter, M., Geron Corporation, 3 (Pos-L186)Cherif-Sheikh, R., Ipsen Pharma S.A., 3 (Pos-L40)Cho, S., PSIA Inc., 5 (Pos-L165)Choi, S., PSIA Inc., 3 (Pos-L165)Chu, G., Amgen, Inc., 3 (Pos-L13)Costantin, J., Molecular Devices Corporation, 3 (Pos-L171)Coulibaly, S., Amgen, Inc., 3 (Pos-L13)Cozzarelli, N., Lawrence Berkeley National Laboratory (Pos-L180) A part of our experi-mental device that is presented is in a patent process through LBNL.Deechongkit, S., Amgen, Inc., 3 (Pos-L13)Fedida, D., Cardiome, 1, 3 (Pos-L99)Fedorov, D., NAREGI, 2 (Pos-L155)Finkel, A., Molecular Devices Corporation, 3 (Pos-L171)Fukuzawa, K., CREST, 2, FSIS, 2 (Pos-L155)Gill, R., Aurora Biomed Inc, 3 (Pos-L95)Gold, J., Geron Corporation, 3 (Pos-L186)Handran, S., Molecular Devices Corporation, 3 (Pos-L171)Hao, X., Geron Corporation, 3 (Pos-L186)He, J., Geron Corporation, 2 (Pos-L186)Hong, S., Lawrence Berkeley National Laboratory, (Pos-L180) A part of our experimentaldevice that is presented is in a patent process through LBNL.Hughes, J., Molecular Devices Corporation, 3 , (Pos-L171)Humphries, D., Lawrence Berkeley National Laboratory , (Pos-L180) A part of our exper-imental device that is presented is in a patent process through LBNL.Jeon, K., PSIA Inc., 3 , (Pos-L165)Kamp, T., Geron Corporation, 2 , (Pos-L186)Kitaura, K., NAREGI, 2 , (Pos-L155)Komeiji, Y., NAREGI, 2 , (Pos-L155)Lebkowski, J., Geron Corporation, 3 , (Pos-L186)Lee, L., GlaxoSmithKline, 2, Intel, 2 , (Pos-L8)Liang, D., Aurora Bimed Inc, 4 , (Pos-L95)Mitchell, K., OB/Gyn CellSecure , (Pos-L88)Nakano, T., CREST, 2, FSIS, 2 , (Pos-L155)Nikonowicz, E., Rice University, 2, 3 , (Pos-L46)Police, S., Geron Corporation, 3 , (Pos-L186)Smith-Maxwell, C., Molecular Devices Corporation, 3 , (Pos-L171)Tanaka, S., CREST, 2 , (Pos-L155)Tworowska, I., Rice University, 3 , (Pos-L46)Valery, C., Ipsen Pharma S.A., 3 , (Pos-L40)Wicks, D., Aurora Biomed Inc, 3 , (Pos-L95)Wittel, A., Molecular Devices Corporation, 3 , (Pos-L171)Xu, C., Geron Corporation, 3 , (Pos-L186)Yang, N., Molecular Devices Corporation, 3 , (Pos-L171)

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