Elucidating Structure and Function of Elucidating Structure and Function of ProteinsProteins
Gerald Kada Ph DGerald Kada, Ph.D.Agilent Technologies (Nano Measurements ( a o easu e e ts
Division)Austria / USA
Dr. Gerald KadaNanoMeasurements Div
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The main Species in BiologyThe main Species in Biology Others:-Lipids-Carbohydrates-Small molecules(ions etc )
From DNA to
(ions etc.)
Proteins
Proteins
DNA (mostly double stranded),RNA (mostly single stranded; viral, siRNA, mRNA, tRNA etc)
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( y g )
Structure Analysis: Live Human Rhinovirus protein shell
Watching RNA release from virus by decreasing pH
Kienberger, Hinterdorfer et al, J Virology (2004)& Structure (2005)
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Structure Analysis: Antibody binding to Protein crystal(Purple membrane) in buffer solution
200 nm 40 nm
0 nm
90 x
90
4 nm
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Kienberger et al, EMBO Reports (2004)
Ultimate Resolution: Higher Harmonics imaging Ultimate Resolution: Higher Harmonics imaging Bacterial SBacterial S--Layer protein crystalLayer protein crystal
Topography 2nd Harmonic ampl.
tude
(nm
) Tip resonance peak
2nd harmonicsFreeze-etchingpreparations
Am
plit
Frequency (kHz)
• <1 nm resolution on protein crystal
Preiner et al, PRL 2007Averaged image of 55 unit cells
<1 nm resolution on protein crystalin liquid (MAC mode)
• 3 pm amplitude sensitivity in 2nd harm.• quantifiable 2nd harmonics amplitude
ff
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Preiner et al, PRL 2007Sleytr et al, PSS 2001=> stiffness data, Young‘s modulus map
Structure of fragile proteins:Structure of fragile proteins:Amyloid fibril formationAmyloid fibril formationyy(Alzheimer, Parkinson)(Alzheimer, Parkinson)
1 µm
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Knowles et al, Science/PNAS 2007
Using Force Spectroscopy to understandUsing Force Spectroscopy to understandAntibodyAntibody––Antigen Interaction Strength (Function) vs. Antigen Interaction Strength (Function) vs. IntraIntra--molecular Antigen Stability (Structure)molecular Antigen Stability (Structure)IntraIntra molecular Antigen Stability (Structure)molecular Antigen Stability (Structure)
Anti-Sendai Antibody on the Tip
Sendai-loop
Tip, Bacteriorhodopsin on the Surface ( “Purple Membrane”)
Unravelling single Bacteriorhodopsin
Sequence-dependent pulling patternKienberger, Kada et al, JMB 2005
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Sequence-dependent pulling pattern –with and without Sendai-loop
g , ,Preiner et al, BJ 2007
Varying the loading rate for xVarying the loading rate for xßß (binding (binding pocket size), kpocket size), koffoff (kinetic off(kinetic off--rate) and rate) and ∆G ∆G (Energy)(Energy)(Energy)(Energy)
Kinetics
120
Kinetics Thermodynamics/affinity∆G = -RTlnKD
80
90
100
110
120 -------- VLDLR1-8 (x
β = 0.38 nm +- 0.03 , koff = 0.13 sec-1 +- 0.08)
-------- VLDLR1-3 (xβ = 0.87 nm +- 0.13, koff = 0.08 sec-1 +- 0.07)
rce
[pN
]
30
40
50
60
70
bind
ing
For
100.0 1000.0 10000.0
20
30
Unb
Loading Rate [pN/sec]R kl Hi t d f
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F*(r) = kBT/xß • ln(r • xß/ koff • kBT) Rankl, Hinterdorfer, Preiner, Univ LinzBell, Evans, Jarzynski, Hummer ‘78,’93,´06
Close look on modified AFM tip
Fully covered AFM tip
Nä j Ki b Eb Hi t d f U i Li
Single Molecule tethering to AFM tip
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Näreoja, Kienberger, Ebner, Hinterdorfer, Uni LinzKada et al, Nanotoday (2008)
Topography and Topography and RecognitionRecognitionRecognition Recognition
Imaging (TREC)Imaging (TREC)
Topography Recognition
50nm
DNA-protein complexes as stored in the chromosomes (“Chromatin”)
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Wang, Lindsay, ASUChromatin plus enzymes
Recognition imaging: Identifying protein tagsRecognition imaging: Identifying protein tagsProtein crystal (S-Layer w Strep-tagII)
Topo RECognition
Seeing the locationof tagged proteinsof tagged proteinsin a protein crystalusing a sensor onthe tip
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Tang et al, Nano Lett 2008the tip
Review article:Review article:Kada, Kienberger, HinterdorferNanotoday 3 12-19 (2008)Nanotoday 3, 12 19 (2008)
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Finally…Thanks for your attention!y
University of Linz Biophysics Institute
Acknowledgements
- University of Linz, Biophysics InstituteProf. Peter HinterdorferProf. Hermann GruberProf. Gerhard SchützJosef MadlFerry Kienberger (now Agilent Labs)Lilia ChtcheglovaJohannes PreinerJilin TangAndreas Ebner
Contact:[email protected]
www.agilent.com/find/nano
Andreas EbnerChristian Rankl (now Agilent Labs)
- Arizona State University, Biodesign InstituteProf. Stuart LindsayHongda WangA il t T h l i N M t- Agilent Technologies, Nano Measurements DivisionTianwei JingW. Travis JohnsonWenhai Han
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e a aSergei Magonov
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