Force measurements on single moleculesafm4nanomedbio.eu/Data/Sites/1/summer_school_2011/rico... · 2016. 2. 17. · Force measurements on single molecules Felix Rico Institut Curie,

Force measurements on single molecules

Felix Rico

Institut Curie, Paris, France

AFM BioMed Summer School Marcoule 2011

3rd European Summer School on Theory and Practice of AFM in Life

Sciences and Medicine

August 29, 2011

Outline

1.! Introduction

2.! Theory

3.! Force spectroscopy methods

4.! Examples

5.! Other methods

Introduction: why mechanics of proteins?

•! Muscle: titin

Klaus Schulten lab http://www.ks.uiuc.edu

•! Muscle: titin •! Cytoskeletal elements: filamin, actin, talin, spectrin

Cooper GM. The Cell: A Molecular Approach. 2nd edition.

Schwaiger, I., et al. 2004 Nat Struct Mol Biol




del Rio, A., et al. 2009 Science




Johnson, C. P., et al. 2007 Science


Cooper GM. The Cell: A Molecular Approach. 2nd edition. Shimaoka 2003 Nature Reviews



•! Extracellular matrix: fibronectin, collagen •! Membrane proteins: integrins, bacteriorhodopsin



pdb 2at9 Mitsuoka et al. 1999 J Mol Biol

p

cytosolic

extracellular

h!"




•! Antibodies



Receptor/ligand (un)binding

Protein (un)folding

Ion channels activity

Vesicle fusion Free energy

Reaction coordinate

kº ~ exp(-#F0/kBT)

x!"

#F0

Biological processes described by an energy

landscape

Bell 1978 Science

-f#x

f

Bell-Evans theory

Force

ln(k

off)

-f#x

f

Bell-Evans theory

Force

ln(k

off)

Bell 1978 Science

Evans and Ritchie 1997 Biophys J

Bell 1978 Science


-f#x

f(t)

ln(rf)

Bell-Evans theory

-f#x

f(t)

ln(rf)

Bell 1978 Science


Bell-Evans theory

Dudko et al. 2006 Phys Rev Lett

Friddle 2008 Phys Rev Lett

More refined theories

Forc

e

distance

The foce-distance curve

loading

rate

force

contact

setpoint z

Foce-distance curves: calibration

invOLS: voltage to deflection spring constant: deflection to force

Hutter and Bechhoefer 1993, Rev Sci Instr

Spring constant calibration methods

Burnham 2003 Nanotechnol

5-20% uncertainty

Interference, viscous drag!

Huang 2006 Meas. Sci. Technol.

Interference, viscous drag!

Alcaraz et al. 2002 Langmuir Janovjak et al. 2005 Eur Biophys J

V-shaped

rectangular

AFM force measurements: dynamic force spectroscopy

Rel

ativ

e fre

quen

cy

Unbinding force (pN) Loading rate (pN/s)

Forc

e

Distance

37 µm/s

3.7 µm/s

0.37 µm/s

Force (pN)

200 nm 30 pN

ks

16012080400

fr

52±21 pN/s

427±73 pN/s

2.7±0.5 nN/s

Life

tune

(s)

0.01

0.1

1

10

12080400

Unb

indi

ng fo

rce

(pN

)

70

60

50

40

30

20

104 6

1022 4 6103 2 4 6

104

A B C D

Rico, F., et al. 2011 Life at the nanoscale

Bustamante, C., et al. 2000 Nat Rev Mol Cell Biol

Sandal, M., et al. 2006 Polymer

Worm-like chain model

AFM force measurements: dynamic force spectroscopy

Crampton, N. and D. J. Brockwell 2010 Curr. Opin. Struct. Biol.

AFM force measurements: force-clamp

Other techniques: locki-in, laser feedback controlled cantilever! (see refs.)

AFM force measurements: DFS vs. force-clamp

DFS Force-clamp

Time resolution 50-100 "s 3-10 ms

Force resolution 5-10 pN >20 pN (lifetime)

Limitations

Tedious data

processing, viscous drag

Complex setup,

feedback loop response

Advantages Simple setup elegant data

processing

AFM force measurements: experimental conditions

•! Indentation force: 500 pN

•! Contact time: 0-1s

•! Retraction speed: 1 nm/s to 100 "m/s

•! Cantilevers:

•! spring constant: 6 pN/nm-100 pN/nm (Biolevers, microlevers,

OTR4)

•! resonance frequency: >5kHz in liquid

•! tip: unsharpened, radius 10-20 nm

•! Force-clamp forces: 20-300 pN (depending on the unfolding lifetime)

•! Samples:

•! gold surface (to allow cysteines to bind)

•! high protein concentration: 0.1 mg/mL

Example: muscle protein titin

Klaus Schulten lab http://www.ks.uiuc.edu

Titin: first AFM measurements

Rief, M., et al. 1997 Science

Titin: force-clamp measurements

Oberhauser, et al. 2001 PNAS

Titin: deeper insight

Marszalek, P. E., et al. 1999 Nature


Marszalek, P. E., et al. 1999 Nature


Lu, H., et al. 1998 Biophys. J. Klaus Schulten lab http://www.ks.uiuc.edu


Lu, H., et al. 1998 Biophys. J. Klaus Schulten lab http://www.ks.uiuc.edu

References

Bell, G. I. (1978). "Models for Specific Adhesion of Cells to Cells." Science 200(4342): 618-627.

Bustamante, C., J. C. Macosko, et al. (2000). "Grabbing the cat by the tail: manipulating molecules one by one."

Nat Rev Mol Cell Biol 1(2): 130-136.

Butt, H. J. and M. Jaschke (1995). "Calculation of thermal noise in atomic force microscopy." Nanotechnology 6:

1-7.

Burnham, N. A., X. Chen, et al. (2003). "Comparison of calibration methods for atomic-force microscopy cantilevers." Nanotechnology 14(1): 1-6.

Crampton, N., K. Alzahrani, et al. (2011). "Mechanically Unfolding Protein L Using a Laser-Feedback-Controlled Cantilever." Biophysical Journal 100(7): 1800-1809.

Crampton, N. and D. J. Brockwell (2010). "Unravelling the design principles for single protein mechanical strength." Current Opinion in Structural Biology 20(4): 508-517.

del Rio, A., R. Perez-Jimenez, et al. (2009). "Stretching Single Talin Rod Molecules Activates Vinculin Binding."

Science 323(5914): 638-641.

Dudko, O. K., A. E. Filippov, et al. (2003). "Beyond the conventional description of dynamic force spectroscopy

of adhesion bonds." Proc Natl Acad Sci U S A 100(20): 11378-81.

Dudko, O. K., G. Hummer, et al. (2006). "Intrinsic rates and activation free energies from single-molecule pulling experiments." Physical Review Letters 96(10).

Dudko, O. K., G. Hummer, et al. (2008). "Theory, analysis, and interpretation of single-molecule force spectroscopy experiments." Proc Natl Acad Sci U S A 105(41): 15755-60.

Evans, E. and K. Ritchie (1997). "Dynamic strength of molecular adhesion bonds." Biophysical Journal 72(4): 1541-1555.

Friddle, R. W. (2008). "Unified model of dynamic forced barrier crossing in single molecules." Physical Review

Letters 100(13).

Friddle, R. W., P. Podsiadlo, et al. (2008). "Near-equilibrium chemical force microscopy." Journal of Physical

Chemistry C 112(13): 4986-4990.

Hutter, J. L. and J. Bechhoefer (1993). "Calibration of atomic-force microscope tips." Review of Scientific Instruments 64(7): 1868-1873.

References

Johnson, C. P., H.-Y. Tang, et al. (2007). "Forced Unfolding of Proteins Within Cells." Science 317(5838): 663-666.

Kellermayer, M. s. S. Z., S. B. Smith, et al. (1997). "Folding-Unfolding Transitions in Single Titin Molecules Characterized with Laser Tweezers." Science 276(5315): 1112-1116.

Lu, H., B. Isralewitz, et al. (1998). "Unfolding of Titin Immunoglobulin Domains by Steered Molecular Dynamics Simulation." Biophysical Journal 75(2): 662-671.

Marszalek, P. E., H. Lu, et al. (1999). "Mechanical unfolding intermediates in titin modules." Nature 402(6757):

100-103.

Oberhauser, A. F., P. K. Hansma, et al. (2001). "Stepwise unfolding of titin under force-clamp atomic force

microscopy." Proceedings of the National Academy of Sciences of the United States of America 98(2): 468-472.

Rico, F., C. Chu, et al. (2011). "Force-Clamp Measurements of Receptor-Ligand Interactions." Methods in Molecular Biology 736: 331-353.

Rief, M., M. Gautel, et al. (1997). "Reversible unfolding of individual titin immunoglobulin domains by AFM." Science 276(5315): 1109-12.

Sandal, M., F. Grandi, et al. (2006). "Single molecule force spectroscopy discovers mechanochemical switches in biology: The case of the disulfide bond." Polymer 47(7): 2571-2579.

Schlierf, M., F. Berkemeier, et al. (2007). "Direct Observation of Active Protein Folding Using Lock-in Force

Spectroscopy." Biophysical Journal 93(11): 3989-3998.

Schwaiger, I., A. Kardinal, et al. (2004). "A mechanical unfolding intermediate in an actin-crosslinking protein."

Nat Struct Mol Biol 11(1): 81-85.

Stark, R. W., T. Drobek, et al. (2001). "Thermomechanical noise of a free v-shaped cantilever for atomic-force microscopy." Ultramicroscopy 86(1-2): 207-215.

Williams, P. M., S. B. Fowler, et al. (2003). "Hidden complexity in the mechanical properties of titin." Nature 422(6930): 446-449

Documents

Force measurements on single moleculesafm4nanomedbio.eu/Data/Sites/1/summer_school_2011/rico... · 2016. 2. 17. · Force measurements on single molecules Felix Rico Institut Curie,