Modulation of Crystal Growth by the Terminal Sequences of thePrismatic-Associated Asprich Protein

Katya Delak,§,† Jennifer Giocondi,§,‡ Christine Orme,‡ and John Spencer Evans*,†

Laboratory for Chemical Physics, Center for Biomolecular Materials Spectroscopy, New YorkUniVersity, 345 E. 24th Street, Room 1007, New York, New York 10010, and, Department ofChemistry and Materials Science, Lawrence LiVermore National Laboratory,LiVermore, California 94551

ReceiVed April 25, 2008; ReVised Manuscript ReceiVed October 1, 2008

ABSTRACT: The formation of calcite in the mollusk shell prismatic layer requires the participation of various proteins. Recentstudies indicate that the prismatic-associated protein superfamily, Asprich, is capable of in vitro stabilization of amorphous calciumcarbonate (ACC), a precursor phase of prismatic calcite. To learn more about the molecular behavior of Asprich, we performedexperiments on two highly conserved sequences derived from Asprich: Fragment-1, a 48 AA N-terminal cationic-anionic sequence,and Fragment-2, a previously characterized 42 C-terminal AA anionic sequence. SEM analyses reveal that Fragment-1 inducespolycrystalline, radial aggregate assemblies of calcite, with evidence of surface porosities. AFM flow cell experiments demonstratethat Fragment-1 is multifunctional and its mineralization behavior is qualitatively similar to that reported for Fragment-2 except forhillock step kinetics. Surprisingly, when Fragment-1 and Fragment-2 are present together within the same assay, we observe phasestabilization of vaterite on Kevlar substrates and amorphous-appearing islands on calcite substrates. We believe that island formationon the calcite substrate results from the deposition of peptide-mineral clusters onto calcite hillock terrace surfaces. These eventsmay also take place on the Kevlar substrate as well, where either vaterite or calcite form. The most significant feature is that amixture of Fragment-1 + Fragment-2 are required to induce these effects and that the individual sequences themselves do not havethis capability. These results indicate that these conserved terminal Asprich sequences jointly exhibit mineralization behavior (i.e.,phase stabilization) that is qualitatively similar to the parent protein, and, parallels the in vitro findings reported for other calcite andaragonite - associated polypeptide sequences. It is likely that the sequence features of Asprich may be used to design crystal growthmimetics that can modulate crystal growth within the laboratory setting.

Introduction

One of the more interesting aspects of the biomineralizationprocess is the participation of specific mineral regulatoryproteins.1-4 As an example, in some mollusk shells there existtwo layers, nacreous and prismatic, each of which possessdifferent polymorphs of calcium carbonate. It is now becomingclear that mineral formation within the nacreous and prismaticlayers of the mollusk shell involves the participation of distinctproteins with different sequence features.3-6 Recent studies ofnacre (aragonite) associated polypeptides7-10 reveal that thesesequences consist of a mixture of anionic, cationic, polar andhydrophobic sequence regions. Some of these nacre polypeptidesbehave in a multifunctional capacity in vitro, that is, theysimultaneously block and accelerate certain aspects of calciumcarbonate crystal growth,10,11 and/or, induce the formation ofnew mineral deposits.11 Conversely, many prismatic (calcite)associated polypeptide sequences are highly negatively chargedand consist of significant percentages of Asp and Gluresidues.12-14 Recent studies indicate that prismatic - associatedproteins, such as caspartin,15 calprismin,15 and prismalin,16 arecapable of modulation calcium carbonate crystal growth andmorphology. However, compared to nacre-specific sequences,little is known regarding the mineral modification capabilitiesof other prismatic-associated sequences.

Recently, a subfamily of seven proteins associated with theprismatic layer of the bivalve, Atrina rigida, were identified andsequenced.12 These proteins, designated as Asprich “a” through

“g”, are multidomain in nature and consist of C- and N-terminalsequences that are highly conserved witin this superfamily.Recent studies indicate that recombinant Asprich induces andstabilizes amorphous calcium carbonate (ACC) in vitro.12b

Hence, Asprich sequences possess important information withregard to essential features that enable phase stabilization andtransformation to prismatic calcite. To obtain this sequenceinformation, a reductionist approach has been employed to studythese highly conserved Asprich N- and C-terminal sequencesas individual entities. The rationale for studying protein sequencefragments and employing them individually and in a combina-torial fashion are to learn more about mollusk shell sequencesand their effects on mineralization under reproducible conditions.This provides two benefits. First, the molecular behavior of theindividual sequences under controlled conditions can provideclues as to their potential participation and function within theparent protein and how this contrasts with sequences obtainedfrom other mollusk shell proteins. Second, the molecularcharacteristics of these sequences can serve as models fordeveloping proteins or polymers which control or modulatecrystal growth for materials or nanotechnological applications.As an example, the conserved C-terminal 42 amino acid (AA)Asprich sequence (Fragment-2)12,17,18 generates irregular crystalgrowth patterns on calcite crystals17 and forms deposits whichadsorb onto calcite dislocation hillock terraces, resulting inmorphological “sculpting” of calcite into a rounded geometry.18

The mineralization activity of Fragment-2 differs from thatobserved for nacre-specific protein sequences,11 and this dem-onstrates that each shell layer employs unique sequence-specificmechanisms to construct distinct mineralized layers.

In contrast, there is no information available for the N-terminal Asprich sequence region. This sequence is comprised

* To whom correspondence should be addressed. Tel: 2129989605. Fax:2129954087. E-mail: jse1@nyu.edu.

§ Indicates equal contributors to this work.† New York University.‡ Lawrence Livermore National Laboratory.

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10.1021/cg8004294 CCC: $40.75 2008 American Chemical SocietyPublished on Web 11/04/2008

of a cationic 7 AA region and a 100 AA Asp, Glu-rich anionicregion.12 Additionally, we do not know if both terminalsequences can jointly modulate crystal growth in a manner thatmirrors the activity of the Asprich protein. This Report detailsinitial structure-function studies of this N-terminal sequence,and, reports an interesting finding with regard to the potentialsynergy between the N- and C-terminal sequences of Asprichduring in vitro crystal growth. As a starting point, we synthe-sized a 48 AA synthetic polypeptide (Fragment-1) representinga chemically synthesizable portion of the N-terminal portionof Asprich (Figure 1).12 Using SEM, we find that Fragment-1induces the formation of radial polycrystalline clusters of calcitethat feature surface voids/porosities, similar to what has beenpreviously reported for egg shell calcite-associated19-22 andprismatic-associated15 proteins. AFM flow cell studies revealthat Fragment-1 is multifunctional and is qualitatively similarto Fragment-2 in terms of hillock morphology effects, depositformation, and crystal interaction, but diverges from Fragment-2in terms of hillock growth kinetics. Interestingly, a mixture ofFragment-1 and Fragment-2 induces vaterite formation23,24 inour SEM assay systems and round terrace islands in our AFMassay systems, indicating that the integral terminal sequencesof Asprich, like the parent protein itself, are jointly capable ofphase stabilization.

Materials and Methods

Polypeptide Synthesis. Free N-terminal, C-R-amide-capped 48-AAFragment-1 polypeptide was synthesized and purified at the 100micromole level at the Wm. Keck Biotechnology Peptide SynthesisFacility, Yale University, by Dr. Janet Crawford, using protocolsdescribed in our earlier work for other Asprich sequences.17,18 TheN-acetyl-capped, free alpha-carboxyl 42 AA Fragment-2 polypeptidewas synthesized and purified as previously described.17,18 C- andN-Terminal alpha amide capping procedures were performed to simulatepeptide bond attachment.7,17,18 After resin cleavage and reverse-phaseHPLC purification (Waters C-18 column, >95% pure),17,18 theexperimental molecular mass of Fragment-1 was determined by matrixassisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS) to be 5178.1 Da, in agreement with the theoreticalvalue of 5177.2 Da.

In Vitro Kevlar Crystal Growth Assays. We employed a polyimide(Kevlar) assay17 for the nucleation of calcium carbonate crystals inthe presence of Fragments-1, -2, and a 1:1 mol ratio mixture ofFragment-1 + Fragment-2. Total polypeptide assay concentrations were1 × 10-5, 5 × 10-5, and 1 × 10-4 M. Negative control conditionsconsisted of no added peptide. Assay conditions and sample workupfor SEM imaging were conducted as described in our previous Asprich

C-terminal polypeptide studies.17,18 Scanning electron microscopyimaging was conducted using a Hitachi S-3500N SEM microscope at5 kV after thin Au coating of samples. The SEM images presented inthis report are representative of 10-20 different crystals in each assaysample. Cropping of SEM images and adjustment of brightness/darknessand contrast levels were performed using Adobe Photoshop.

Micro-Raman Spectroscopy of Kevlar-Nucleated Crystals. Toverify the formation of calcium carbonate polymorphs in the presenceof Fragment-1 + Fragment-2 mixture, micro-Raman analysis wasperformed on Kevlar-nucleated crystals which exhibited both rhom-bohedral and nonrhombohedral features, and, for comparison, the Kevlarthreads themselves. Micro-Raman spectroscopy measurements weremade with a commercial instrument (Horiba-Jobin Yvon, Edison, NJ)employing a 10 mW 632.8 nm He-Ne laser as the probe beam. Theinstrument settings were as follows: 600 spacing/mm grating, 300micron hole, 100 micron slit, and 20× or 100× microscope objectiveswere used. Each spectrum was obtained by averaging three 60 s runs.

In Situ AFM. AFM experiments were performed on surfaces ofanchored calcite crystals (freshly cleaved geologic calcite from Brazil,e1 mm each dimension) that were subjected to overgrowth via exposureto a supersaturated solution of CaCl2/NaHCO3 (2.5 mM CaCl2, 2.5 mMNaHCO3 in deionized distilled water) and imaged in real time usingan atomic force microscope outfitted with a commercially availablefluid cell (Nanoscope III, Digital Instruments, Santa Barbara, CA).11,18

Prior to peptide introduction into the fluid cell system, each calcitecrystal was equilibrated via exposure to supersaturated solution; thechoice of flow rate (1 mL/min) was such that step kinetics were notlimited by bulk diffusion. Once well-defined hillocks were detected,polypeptides were then introduced using a freshly prepared supersatu-rated solution at the same flowrate. This solution consisted of a givenpeptide dissolved in NaHCO3 solution, which was filtered using a 0.2µM PVDF Gelman Acrodisc11,18 and then mixed with CaCl2 solution,with the final concentration of both CaCl2 and NaHCO3 ) 2.5 mMand the final peptide concentration ) 3, 6, or 12 µM. We also ranparallel imaging experiments on a 1:1 peptide mixture of 3 µMFragment-1 + 3 µM Fragment-2. Negative control conditions utilizedsupersaturated solutions that were devoid of any added peptide; thesesolutions normally have a pH of 8.3, and therefore were adjusted to8.1 by adding a minute amount of dilute aqueous HCl. The addition ofFragment-1 to the unadjusted negative control solution (pH ) 8.3) ledto an approximate downshift to pH 7.3, which was then adjusted topH 8.1 with minute volumes of KOH to avoid downshift in supersatu-ration or slowing of step growth kinetics. The supersaturation ratio S) [{Ca2+}{CO3

2-}/Ksp,calcite] and the activity ratio {Ca2+}/(CO32-}

of pure solutions (no polypeptide) at pH 8.1 were computed usingGeochemists’ Workbench software (v.5, Rockware, Inc.) and found tobe 6.1 and 130, respectively. The chelation of calcium ions by thepeptide is not expected to shift the supersaturation appreciably due tothe low peptide/Ca(II) ratio. All imaging was performed on the {104}cleavage plane of calcite.

AFM fluid cell imaging was performed at room temperature withimage collection commencing 5 min after the introduction of eachpolypeptide.11,18 In situ images were collected in contact mode usingSi3N4 tips and were limited to regions undergoing step growth atdislocation hillocks of calcite. The imaging force was reduced to theminimum possible value that allowed the tip to remain in contact withthe surface, such that there was no measurable effect on the growthkinetics.11,18 AFM imaging was typically performed once with a givenpolypeptide sample, and no local erosion or local enhancement of stepvelocities was observed due to tip effects. Note that step-angle distortionexists in the images because the step front advances during the scantime. Images reported here are not corrected for this effect.11,18 Allimages were processed with Image SXM (version 174-1X) for color,brightness, contrast adjustment, and examination of height profile. Dryimaging of calcite crystals exposed to Fragment-1 were obtained byfirst removing the crystal from the fluid cell, rinsing the crystal with10 mL of deionized distilled water and drying with clean compressednitrogen gas. Samples were then imaged in tapping mode. Final imageadjustment for all samples was made using Adobe Photoshop.

In addition to the AFM studies described above, we conductedparallel AFM imaging studies of calcite dislocation hillock growth inthe presence of a 1:1 peptide mixture composed of 50 µM Fragment-1+ 50 µM Fragment-2, over a total period of 18 h. Note that these higherconcentration studies were performed with some modifications toprovide a comparison with our Kevlar nucleation assays described in

Figure 1. Primary sequence of the conserved N-terminal region ofAsprich (Fragment-1). Numbering system (black) corresponds to thecleaved, mature polypeptide, minus the N-terminal signal peptideregion.15 Original numbering system12 is shown above Fragment-1 inparentheses. Fragment-2 domain of Asprich is shown for comparison.

4482 Crystal Growth & Design, Vol. 8, No. 12, 2008 Delak et al.

the preceding sections. A 10 mL solution was used to flow throughthe imaging flow cell at a slower rate (e0.5 mL/min) with recirculationof the solution to extend the experiment to longer time intervals thatmatched those of the Kevlar assay study (i.e., 18 h). Thus, these long-term studies are expected to experience concentration and pH variationsover time; in fact, the final pH of the solution at the conclusion of theexperiment was found to be ∼8.9.

CD Spectrometry. Circular dichroism (CD) spectra were obtainedfor Fragment-1 and Fragment-2 at 20 °C, using an AVIV 60 CDspectrometer (60DS software version 4.1t). The CD spectrometer waspreviously calibrated with d-10-camphorsulfonic acid. The peptidesamples (apo form) were dissolved and diluted to final concentrationsof 8 µM in 100 µM Tris HCl (pH 7.5). For all spectra, wavelengthscans were conducted from 185 to 260 nm with appropriate backgroundbuffer and CaCl2 subtraction, using a total of four scans with 1 nmbandwidth and 0.5 nm/s scan rate17,18 In all CD spectra, mean residueellipticity [θM] is expressed in deg · cm2 · dmol-1.17,18

Results and Discussion

Fragment-1 Modulation of Calcite Crystal Morphologyand Kinetics. Fragment-1 induces the growth of micron-sizedspherical assemblies or clustered arrays of polycrystalline calciteclusters on Kevlar threads (Figure 2). We also note the presenceof voids or porosities on the surface of calcite crystals grownin the presence of Fragment-1 (Figure 2D, note arrows). Notethat these surface features have been observed in crystal growthassays containing egg shell-associated polypeptides.19-22 Thisphenomenon was also reported for Asprich Fragment-2,17 andwe believe that these surface voids arise from peptide-mineralinteractions that lead to uneven crystal growth rates and“fingering” effects,17 as we note below in our AFM studies.

Interestingly, the spherical or radial assemblies observed inFigure 2 are composed of small rhombohedral crystals thatfeature gaps or spacings between crystals. There may be oneor more underlying mechanisms which would explain theformation of crystal assemblies with these features. For example,Fragment-1-induced crystal twinning may be occurring (Figure2B,C), as evidenced by the presence of gaps or spaces betweenadjacent calcite rhombohedral crystals (note arrows in Figure2B).15 Note that the biological twinning phenomenon has beenreported for calcite crystals growth in the presence of prismatic-

associated proteins.15b However, since X-ray diffraction tech-niques have not been applied to the crystals in our assays, thereis no physical evidence to verify crystal twinning. Alternatively,other events may have led to the formation of the gaps, spacings,or radial orientations of the aggregate crystals, such as a gradualshift in crystal orientation across the aggregate in response toshrinkage stresses induced by ACC transformation.12,24 Al-though the true mechanism of crystal aggregate formation isnot known, the fact that Fragment-1 generates these clustersindicates that this sequence is controlling mineralization eventseither at the mineral interface or in solution, ultimately affectingcalcite crystal morphology in this unique fashion.

Using AFM imaging, we note that the introduction ofFragment-1 produces several significant effects on calcitedislocation hillock morphology that are shared with thoseproduced by Fragment-2 (Figure 3).18 First, we note thatFragment-1, like Fragment-2, induces the formation of clusterson terrace surfaces, and we believe that these clusters representpeptide-mineral aggregates or deposits.11,18 Interestingly, at lowpolypeptide concentrations, steps continue to grow under theseclusters. Second, the morphology of acute-obtuse corner sitesbecomes rounded, losing the sharp delineation between acuteandobtusestepdirections,leadingtoanoverallovalmorphology.11,18

Third, we observe acute step “bunching” as noted by theappearance of roughened acute step edges that are not observedin the negative control scenario. Fourth, we observe that the

Figure 2. Scanning electron microscopy images of in vitro Kevlarcalcium carbonate assay systems. (A) negative control assay, whichfeatures typical rhombohedral calcite crystals; (B) Fragment-1, 50 µM.Here, arrows denote gaps between crystals, presumable arising fromcrystal twinning; (C) Fragment-1, 100 µM; (D) as per (C), but at highermagnification, revealing the presence of surface porosities (arrows).Scalebars indicate image dimensions. Figure 3. AFM contact mode images of the dislocation hillock region

of calcite crystals. (A) Fragment-1, 6 µM; (B) Fragment-1, 12 µM;(C) dry topographic image of hillock terrace region after exposure to6 µM Fragment-1. All images are shown in the same orientation withthe two acute steps at the top of the images. AFM inset images arethose recorded immediately prior to the introduction of Fragment-1(i.e., negative control conditions). Scalebars indicate image dimensions.Atomic force microscopy on the {104} cleavage plane of calcite revealsthat dislocation hillock growth proceeds via atomic step advancementsover the range of conditions used in these experiments. The atomicstep directions reflect typical rhombohedral calcite crystal morphologywith step risers along four of the six crystallographically equivalent{104} facets (A). Two of the steps are typically termed “acute” (topportion of A) due to the acute angle that the step riser makes withrespect to the cleavage plane, and two of the steps are termed “obtuse”(bottom portion of A) due to the obtuse angle the step riser makeswith respect to the cleavage plane.11,18

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obtuse steps become very rough over time with a “finger”geometry that can be distinguished from classically scallopedpinning morphologies in the following way. This fingermorphology (and therefore the step kinetics) is anisotropic withlong, narrow peninsulas growing through blocked regions. Thisanisotropy is not typical of classical step-pinning modelsalthough curvature controlled effects may also occur. The ap-pearance of a finger is consistent with limited kink mobilityalong the step so that growth occurs via local attachment atregions that are not blocked by impurities. The sides of thesepeninsulas grow slowly or not at all so that they maintain ahigh aspect ratio. Interestingly, finger regions have also beenobserved in AFM experiments with Fragment-2, where thefingering breaks up the steps, creating a porous surface (see forexample Figure 3C).18 It is presumed that the porosities observedon macroscopic calcite crystals (Figure 2D) arise from thisfingering process.

Kinetic measurements (Supporting Information, Figure S1)indicate that obtuse step velocities are higher in the presenceof Fragment-1 (as compared to peptide-free solutions) andremain relatively constant when plotted against step distancefrom the dislocation source. In contrast, earlier experimentsconducted with the Asprich Fragment-2 indicated that thissequence induces initial velocity acceleration followed bydeceleration due to pinning.18 In conclusion, although themineral recognition properties, deposit formation, finger forma-tion and induced hillock morphologies of Fragment-1 andFragment-2 are qualitatively similar, we find that both sequencesdiffer in terms of hillock growth kinetics. We believe that thisdifference in kinetics reflects the different modes that eachsequence utilizes to bind and pin obtuse steps.

Structural features of Fragment-1. The conformation ofFragment-1 was qualitatively investigated using CD spectrom-etry (Figure 4) and compared against data previously obtainedfor Fragment-2.17,18 Here, we note that apo-Fragment-1 isqualitatitively similar to Fragment-2: both sequences exhibit amajor (-) ellipticity band (π-π* transition) at 198 nm, corre-sponding to random coil or unstructured conformation.11,17,18

However, Fragment-1 also possesses a minor, (+) band (n-π*transition) centered near 218 nm, corresponding to polyprolineType II (PPII) extended helical conformation.17,18 Given thatthe 7 AA cationic region of Fragment-1 possesses a Pro residueand three positively charged residues (Figure 1), we infer thatthe PPII-like characteristics of Fragment-1 may arise from thesecompositional features.25 Thus, although Fragment-2 and Frag-ment-1 are qualitatively similar in structure, one structural traitis unique to Fragment-1, and that is the presence of PPII

structure. At this time we do not know what region(s) of eithersequence contain PPII structure or how this structure plays arole in the in vitro mineralization events noted in our report.

Crystal Growth Behavior in the Presence of Frag-ment-1 and Fragment-2 Polypeptides. In our previous studies,it was noted that a 1:1 mixture of the two subdomains thatcomprise Fragment-2 alters crystal growth in a manner thatdiffers from the mineralization effects noted for Fragment-2 andfor the individual subdomains themselves.17,18 To extend thisobservation further, we ran parallel Kevlar mineralization assaysusing a 1:1 mol ratio of Fragment-1 and Fragment-2 (Figure5). Here, we note that the formation of rhombohedral calcite(Figure 5A,C) is also accompanied by the formation ofspherulitic clusters that resemble vaterite (representing ap-proximately 10-30% of the total crystals formed, Figure 5B,D).Note that the formation of vaterite was not observed in thepresence of Fragment-1 alone (Figure 2) or in our previousstudies with Fragment-2 alone.18

Given the small sizes and limited numbers of crystals grownin our Kevlar assays, the use of bulk characterization techniques,such as X-ray diffraction, are seriously limited for distinguishingdifferent polymorph structures. However, techniques such asmicro-Raman spectroscopy can be employed to focus on

Figure 4. Far UV CD spectra of Fragment-1, Fragment-2 at pH 7.5 inthe presence of 100 µM Tris-HCl buffer.

Figure 5. Visual and spectroscopic analyses of crystal growth in thepresence of Fragment-1 and Fragment-2. (A) SEM image of calcitecrystals obtained from in vitro Kevlar calcium carbonate negativecontrol assay; (B) SEM image of calcium carbonate crystals grown inthe presence of 1:1 mol mixture (total peptide concentration ) 100µM) of Fragment-1 + Fragment-2. Scalebar ) 10 microns. Notevaterite-type morphology. (C) and (D) are light microscope images ofa rhombohedral calcite (C) and vaterite-type crystals (D) obtained from1:1 mixture assay and utilized for microRaman analysis. (E) Ramananalysis of “C” and “D” crystals along with the experimental spectraof the Kevlar fibers and the literature values from reference for calcite(blue) and vaterite (red), shown as bars along the bottom of this graphfor comparison. The vertical axis is intensity in arbitary units. TheRaman modes for both calcite and vaterite can be found in Table S1(Supporting Information).

4484 Crystal Growth & Design, Vol. 8, No. 12, 2008 Delak et al.

individual crystals and verify their lattice structures. Usingmicro-Raman spectroscopy, we confirmed that the spheruliticclusters are vaterite (Figure 5E) via comparison to spectraobtained for calcium carbonate polymorphs (Supporting Infor-mation, Table S1). The two key areas of comparison are thelattice modes at low wavenumbers and symmetric stretch (η1)mode around 1085 cm-1. For vaterite, there is a triplet in thelattice mode region and a split peak in the symmetric stretchregion that distinguishes it from both calcite and aragonite. Asexpected, the negative control crystal exhibits the typicalrhombohedral shape of calcite and the micro-Raman spectrumconfirms this assignment (Figure 5). We did not detect thepresence of ACC in these assays; however, given that Fragment1 generates radial crystal aggregates (Figure 2), and, thatspherulitic vaterite crystals are forming in our assays (Figure5), it is possible that an amorphous calcium carbonate phasewas initially present prior to the appearance of calcite or vaterite.

To further investigate this interesting finding, we utilizedAFM to monitor calcite hillock growth and kinetics in thepresence of a 1:1 Fragment-1/Fragment-2 mixture (Figure 6)as a function of time. After 20 min of exposure, we observehillock features that are associated with the presence of eitherFragment-1 or Fragment-2: the formation of clusters on terracesurfaces, the roundening of acute-obtuse corner regions and thefingering of the obtuse steps that leads to porosity formation.Simulatenously, a new feature begins to emerge, namely, theformation of two-dimensional round islands on hillock surfaces.As time evolves, the predominant growth mechanism changesfrom step growth to nucleation and spreading of two-dimensional islands and the formation of clusters on hillocksurfaces (Figure 6). Hence, the 1:1 mixture leads to the inductionof morphological features and crystal growth mechanisms thatare atypical for the individual fragments.

We repeated these AFM flowcell experiments at higherpeptide concentrations (i.e., 100 µM, Supporting Information,

Figure S2) utilized for the Kevlar assays. Under these conditions,we observe two competing processes taking place on the teraces:the formation of etch pits and the simultaenous nucleation oftwo-dimensional round islands. Due to a several minute gap inimaging after new solutions were introduced into the fluid cell,we cannot be certain that the pits represent the active dissolutionof calcite, or, peptide induced “dead” spots on the crystal thatmaterial grows around thereby creating a hole. Nonetheless itis clear that the growth mode has changed and that the newgrowth does not have facets and that pit formation ceases afterthe new overlayer has grown. Using Raman spectroscopy, wewere unable to detect the presence of noncalcite calciumcarbonate polymorphs in this new overgrowth layer (data notshown). Given that the forming layer may not be sufficientlythick to be detected against the larger calcite background, or,that assay solution conditions may have led to calcite transfor-mation over time, we cannot identify the true mineral phase ofthese islands.

We believe that island formation on the calcite substrateresults from the deposition of peptide-mineral clusters ontoterrace surfaces, which are observed in our AFM experimentswith individual Fragment-218 and Fragment-1 (Figure 3) pep-tides. Once these deposits are on the terrace surfaces, furthernucleation events occur at these regions, possibly involving thetransient formation of ACC, since this would be consistent withthe rounded island appearance. This, in turn, rapidly crystallizesinto calcite on the calcite substrate (Figure 6). These eventsmay also take place on the Kevlar substrate as well (Figure 5),where either vaterite or calcite form. Regardless of the substrate,the most significant feature is that a mixture of Fragment-1 +Fragment-2 are required to induce these effects (Figures 5 and6), and that the individual sequences themselves do not havethis capability.

In summary, the N- and C-terminal domains of the Asprichprotein superfamily are multifunctional and can induce clusterdeposits on calcite terraces, pin steps, and affect calcite hillockmorphology or shape in similar ways (Figure 3). In addition,these two conserved sequence domains functionally diverge ina number of important areas. First, only Fragment-1 can inducepolycrystalline aggregation (Figure 2). The fact that crystalaggregates form with radial orientations and with gaps or spacesbetween crystals suggests a number of possible scenarios, suchas twinning,15 or shifts in crystal orientation or shrinkage stressesarising from the transformation of ACC12,24 at some point duringthe assay period. These issues will be followed up in asubsequent report. Second, each sequence exhibits slightlydifferent propensities for obtuse step-pinning which causesdifferences in the step growth kinetics (Supporting Information18

What molecular feature(s) could give rise to these moleculardifferences? One obvious starting point would be the unique 7AA cationic region within the Fragment-1 sequence. Here, it islikely that the presence of Pro and a short polyelectrolyte clusterregion induce some degree of PPII structure25 that we detect inour CD experiments (Figure 4A). We believe that this cationicregion influences Fragment-1 to form clusters or deposits and/or interact with specific calcite hillock features (Figure 3) which,in turn, might influence morphology in a manner that is distinctfrom that of Fragment-2. Another sequence feature whichdistinguishes Fragment-1 from Fragment-2 is the Asp and Gluresidue content and the corresponding net electrostatic charge.In Fragment-1, 31% of the sequence is Asp, 13% of thesequence is Glu, and the ratio of Asp/Glu ) 2.5; the net chargeof this sequence ) -17 at neutral pH.26 In Fragment-2, 36%of the sequence is Asp, 20% of the sequence is Glu, the Asp/

Figure 6. AFM contact mode images of the dislocation hillock regionof calcite crystals in the presence of 1:1 mol mixture of Fragment-1 +Fragment-2. Image (A) represents the negative control assay; Images(B), (C), and (D) are taken in the presence of 3 µM Fragment-1 + 3µM Fragment-2 at t ) 20, 199, and 211 min from the time of peptideaddition, respectively. Image dimensions: A, B, C ) 15 × 15 µm, D) 8 × 8 µm.

Crystal Growth Modulation by Asprich Protein Crystal Growth & Design, Vol. 8, No. 12, 2008 4485

Glu ratio ) 1.9, and the net charge ) -24.26 Hence, aminoacid composition, backbone structure, and electrostatics mayjointly contribute to the molecular differences that we observein vitro.

The most striking finding of our study was the stabilizationof vaterite (Figure 5) and formation of amorphous or round-appearing islands (Figure 6) on calcite substrates in the presenceof Fragment-1 and Fragment-2. These findings suggest thatFragment-1 and Fragment-2 jointly exhibit phase stabilizationproperties, and we note that phase stabilization (i.e., ACC) is areported feature of the parent protein, Asprich.12 As a conse-quence, we believe that the in vitro mineralization behavior ofFragment-1 and Fragment-2 is quallitatively similar to that ofAsprich itself. Although the true identity of calcium carbonatephase(s) within these islands is unknown (Figure 6), their roundappearance and the fact that Fragment-1 and Fragment-2individually induce amorphous deposits upon terrace surfaces(Figure 3)18 suggests that at some point ACC formation ornucleation occurs on hillock surfaces. At present, we do notknow the mechanism(s) by which Fragment-1 + Fragment-2stabilize these phases, nor do we know what percentage of eitherfragment sequence interacts or participates in the formation ofnoncalcitic phases within our assays. However, it has beensuggested that protein stabilization of spherulitic vaterite or ACCarises from polypeptide carboxylate interactions with the mineralphase.12,23 If true, then it is likely that the high carboxylate Asp,Glu content in both Asprich terminal sequences exerts someeffect on polypeptide-mineral, polypeptide-Ca (II), or evenpolypeptide-polypeptide complexation and assembly. In turn,one or more of these molecular phenomena facilitate phasestabilization. Obviously, additional studies will be required toexplore these potential mechanisms and synergistic interactionsbetween Fragment-1 and Fragment-2 and compare these to whattakes place within the Asprich protein itself. The fact that anionicprotein sequences can stabilize phases such as vaterite or ACCmay prove to be a useful model for developing polypeptide-based phase transformation and crystal engineering techniquesin the laboratory for materials and nanotechnology applications.

Acknowledgment. This work was supported by funding fromthe National Science Foundation (DMR-0704148, to J.S.E.).Portions of this work performed under the auspices of the U.S.Department of Energy by Lawrence Livermore National Labo-ratory under Contract DE-AC52-07NA27344. This paper rep-resents contribution number 46 from the Laboratory forChemical Physics, New York University.

Supporting Information Available: Step velocity plot (Figure S1),AFM imaging of hillock regions in the presence of 100 µM peptideconcentrations (Figure S2), and table of Raman wavenumbers andmodes for calcium carbonates (Table S1). This material is availablefree of charge via the Internet at http://pubs.acs.org.

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(26) These charge calculations take into account the presence of C-terminalamide capping, and, the Arg, Lys charge contributions arising fromthe 7 AA catioinic sequence of Fragment-1.

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