Skip to main content
SpringerLink
Log in
Book cover

Atomic Force Microscopy in Biomedical Research pp 331–353Cite as

Force-Clamp Measurements of Receptor–Ligand Interactions

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 736))

Abstract

Protein–protein interactions are the basis of both biochemical and biophysical signaling of living cells. In many cases, the receptor is present on the cell surface while the ligand is in solution or linked to another support (extracellular matrix or another cell). In the case of cellular adhesion, forces are continuously applied to receptor–ligand complexes and, as a consequence, the dissociation kinetics of the bonds may change. It is, thus, relevant to study the kinetics of protein–protein interactions in response to applied forces, as this is the most physiologically relevant situation. The atomic force microscope (AFM) was one of the first nanotools to be applied to this end. However, new approaches need to be developed to better understand the complex energy landscape of molecular interactions under applied stress. In this chapter, we described the use of the AFM to carry out force-clamp measurements on receptor–ligand bonds. Force-clamp measurements on bonds consist of applying a constant and controlled force to a receptor–ligand bond and measure the resulting dissociation lifetime. The described methods include the required materials, functionalization of tips and substrates, force-clamping measurements, and processing and interpretation of the results. An illustrative example is given with the well-studied streptavidin–biotin complex.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   179.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Bell, G. I. (1978) Models for Specific Adhesion of Cells to Cells, Science 200, 618–627.

    Article  PubMed  CAS  Google Scholar 

  2. Moy, V. T., Florin, E. L., and Gaub, H. E. (1994) Intermolecular Forces and Energies Between Ligands and Receptors, Science 266, 257–259.

    Article  PubMed  CAS  Google Scholar 

  3. Florin, E. L., Moy, V. T., and Gaub, H. E. (1994) Adhesion Forces Between Individual Ligand-Receptor Pairs, Science 264, 415–417.

    Article  PubMed  CAS  Google Scholar 

  4. Lee, G. U., Kidwell, D. A., and Colton, R. J. (1994) Sensing Discrete Streptavidin Biotin Interactions with Atomic-Force Microscopy, Langmuir 10, 354–357.

    Article  CAS  Google Scholar 

  5. Evans, E., and Ritchie, K. (1997) Dynamic strength of molecular adhesion bonds, Biophys. J. 72, 1541–1555.

    Article  PubMed  CAS  Google Scholar 

  6. Benoit, M., Gabriel, D., Gerisch, G., and Gaub, H. E. (2000) Discrete interactions in cell adhesion measured by single-molecule force spectroscopy, Nature Cell Biology 2, 313–317.

    Article  PubMed  CAS  Google Scholar 

  7. Zhang, X. H., Wojcikiewicz, E., and Moy, V. T. (2002) Force spectroscopy of the leukocyte function-associated antigen-1/intercellular adhesion molecule-1 interaction, Biophysical Journal 83, 2270–2279.

    Article  PubMed  CAS  Google Scholar 

  8. Hinterdorfer, P., Baumgartner, W., Gruber, H. J., Schilcher, K., and Schindler, H. (1996) Detection and localization of individual antibody-antigen recognition events by atomic force microscopy, Proc Natl Acad Sci U S A 93, 3477–3481.

    Article  PubMed  CAS  Google Scholar 

  9. Dufrene, Y. F., and Hinterdorfer, P. (2008) Recent progress in AFM molecular recognition studies, Pflugers Arch 456, 237–245.

    Article  PubMed  CAS  Google Scholar 

  10. Muller, D. J., Helenius, J., Alsteens, D., and Dufrene, Y. F. (2009) Force probing surfaces of living cells to molecular resolution, Nat Chem Biol 5, 383–390.

    Article  PubMed  Google Scholar 

  11. Merkel, R., Nassoy, P., Leung, A., Ritchie, K., and Evans, E. (1999) Energy landscapes of receptor-ligand bonds explored with dynamic force spectroscopy, Nature 397, 50–53.

    Article  PubMed  CAS  Google Scholar 

  12. Li, F. Y., Redick, S. D., Erickson, H. P., and Moy, V. T. (2003) Force measurements of the alpha(5)beta(1) integrin-fibronectin interaction, Biophys.J. 84, 1252–1262.

    Article  PubMed  CAS  Google Scholar 

  13. Dudko, O. K., Hummer, G., and Szabo, A. (2006) Intrinsic rates and activation free energies from single-molecule pulling experiments, Phys.Rev.Lett. 96.

    Google Scholar 

  14. Hummer, G., and Szabo, A. (2001) Free energy reconstruction from nonequilibrium single-molecule pulling experiments, Proc Natl Acad Sci U S A 98, 3658–3661.

    Article  PubMed  CAS  Google Scholar 

  15. Dudko, O. K., Hummer, G., and Szabo, A. (2008) Theory, analysis, and interpretation of single-molecule force spectroscopy experiments, Proc Natl Acad Sci U S A 105, 15755–15760.

    Article  PubMed  CAS  Google Scholar 

  16. Freund, L. B. (2009) Characterizing the resistance generated by a molecular bond as it is forcibly separated, Proceedings of the National Academy of Sciences 106, 8818–8823.

    Article  CAS  Google Scholar 

  17. Sulchek, T. A., Friddle, R. W., Langry, K., Lau, E. Y., Albrecht, H., Ratto, T. V., DeNardo, S. J., Colvin, M. E., and Noy, A. (2005) Dynamic force spectroscopy of parallel individual Mucin1-antibody bonds, Proc.Natl.Acad.Sci.U.S.A. 102, 16638–16643.

    Article  PubMed  CAS  Google Scholar 

  18. Oberhauser, A. F., Hansma, P. K., Carrion-Vazquez, M., and Fernandez, J. M. (2001) Stepwise unfolding of titin under force-clamp atomic force microscopy, Proc. Natl. Acad. Sci. USA 98, 468–472.

    Article  PubMed  CAS  Google Scholar 

  19. Marshall, B. T., Long, M., Piper, J. W., Yago, T., McEver, R. P., and Zhu, C. (2003) Direct observation of catch bonds involving cell-adhesion molecules, Nature 423, 190–193.

    Article  PubMed  CAS  Google Scholar 

  20. Marshall, B. T., Sarangapani, K. K., Lou, J. H., McEver, R. P., and Zhu, C. (2005) Force history dependence of receptor-ligand dissociation, Biophys. J. 88, 1458–1466.

    Article  PubMed  CAS  Google Scholar 

  21. Pincet, F., and Husson, J. (2005) The solution to the streptavidin-biotin paradox: The influence of history on the strength of single molecular bonds, Biophys.J. 89, 4374–4381.

    Article  PubMed  CAS  Google Scholar 

  22. Rico, F., and Moy, V. T. (2007) Energy landscape roughness of the streptavidin-biotin interaction, J Mol Recognit 20, 495–501.

    Article  PubMed  CAS  Google Scholar 

  23. Butt, H. J., Cappella, B., and Kappl, M. (2005) Force measurements with the atomic force microscope: Technique, interpretation and applications, Surface Science Reports 59, 1–152.

    Article  CAS  Google Scholar 

  24. Weisenhorn, A. L., Maivald, P., Butt, H. J., and Hansma, P. K. (1992) Measuring Adhesion, Attraction, And Repulsion Between Surfaces In Liquids With An Atomic-Force Microscope, Phys. Rev. B 45, 11226–11232.

    Article  Google Scholar 

  25. Proksch, R., Schaffer, T. E., Cleveland, J. P., Callahan, R. C., and Viani, M. B. (2004) Finite optical spot size and position corrections in thermal spring constant calibration, Nanotechnology 15, 1344–1350.

    Article  Google Scholar 

  26. Devasia, S., Eleftheriou, E., and Moheimani, S. O. R. (2007) A survey of control issues in nanopositioning, Ieee Transactions on Control Systems Technology 15, 802–823.

    Article  Google Scholar 

  27. Neuert, G., Albrecht, C., Pamir, E., and Gaub, H. E. (2006) Dynamic force spectroscopy of the digoxigenin-antibody complex, Febs Letters 580, 505–509.

    Article  PubMed  CAS  Google Scholar 

  28. Odorico, M., Teulon, J. M., Bessou, T., Vidaud, C., Bellanger, L., Chen, S. W. W., Quemeneur, E., Parot, P., and Pellequer, J. L. (2007) Energy landscape of chelated uranyl: Antibody interactions by dynamic force spectroscopy, Biophysical Journal 93, 645–654.

    Article  PubMed  CAS  Google Scholar 

  29. Ray, C., Brown, J. R., and Akhremitchev, B. B. (2007) Correction of systematic errors in single-molecule force spectroscopy with polymeric tethers by atomic force microscopy, Journal of Physical Chemistry B 111, 1963–1974.

    Article  CAS  Google Scholar 

  30. Francius, G., Alsteens, D., Dupres, V., Lebeer, S., De Keersmaecker, S., Vanderleyden, J., Gruber, H. J., and Dufrene, Y. F. (2009) Stretching polysaccharides on live cells using single molecule force spectroscopy, Nature Protocols 4, 939–946.

    Article  PubMed  CAS  Google Scholar 

  31. Gittes, F., and Schmidt, C. F. (1998) Thermal noise limitations on micromechanical experiments, European Biophysics Journal with Biophysics Letters 27, 75–81.

    Article  CAS  Google Scholar 

  32. Noy, A. (2008) Strength in numbers: Probing and understanding intermolecular bonding with chemical force microscopy, Scanning 30, 96–105.

    Article  PubMed  CAS  Google Scholar 

  33. Walton, E. B., Lee, S., and Van Vliet, K. J. (2008) Extending Bell’s model: How force transducer stiffness alters measured unbinding forces and kinetics of molecular complexes, Biophys. J. 94, 2621–2630.

    Article  PubMed  CAS  Google Scholar 

  34. Tshiprut, Z., Klafter, J., and Urbakh, M. (2008) Single-molecule pulling experiments: when the stiffness of the pulling device matters, Biophys J 95, L42–44.

    Article  PubMed  CAS  Google Scholar 

  35. Roters, A., and Johannsmann, D. (1996) Distance-dependent noise measurements in scanning force microscopy, Journal of Physics-Condensed Matter 8, 7561–7577.

    Article  CAS  Google Scholar 

  36. Alcaraz, J., Buscemi, L., Puig-de-Morales, M., Colchero, J., Baro, A., and Navajas, D. (2002) Correction of microrheological measurements of soft samples with atomic force microscopy for the hydrodynamic drag on the cantilever, Langmuir 18, 716–721.

    Article  CAS  Google Scholar 

  37. Viani, M. B., Schaffer, T. E., Chand, A., Rief, M., Gaub, H. E., and Hansma, P. K. (1999) Small cantilevers for force spectroscopy of single molecules, Journal of Applied Physics 86, 2258–2262.

    Article  CAS  Google Scholar 

  38. Junker, J. P., Ziegler, F., and Rief, M. (2009) Ligand-Dependent Equilibrium Fluctuations of Single Calmodulin Molecules, Science 323, 633–637.

    Article  PubMed  CAS  Google Scholar 

  39. Berquand, A., Xia, N., Castner, D. G., Clare, B. H., Abbott, N. L., Dupres, V., Adriaensen, Y., and Dufrene, Y. F. (2005) Antigen binding forces of single antilysozyme Fv fragments explored by atomic force microscopy, Langmuir 21, 5517–5523.

    Article  PubMed  CAS  Google Scholar 

  40. Brewer, N. J., Janusz, S., Critchley, K., Evans, S. D., and Leggett, G. J. (2005) Photooxidation of self-assembled monolayers by exposure to light of wavelength 254 nm: A static SIMS study, Journal of Physical Chemistry B 109, 11247–11256.

    Article  CAS  Google Scholar 

  41. Ebner, A., Wildling, L., Kamruzzahan, A. S. M., Rankl, C., Wruss, J., Hahn, C. D., Holzl, M., Zhu, R., Kienberger, F., Blaas, D., Hinterdorfer, P., and Gruber, H. J. (2007) A new, simple method for linking of antibodies to atomic force microscopy tips, Bioconjugate Chemistry 18, 1176–1184.

    Article  PubMed  CAS  Google Scholar 

  42. Florin, E. L., Moy, V. T., and Gaub, H. E. (1994) Adhesion forces between individual ligand-receptor pairs, Science 264, 415–417.

    Article  PubMed  CAS  Google Scholar 

  43. Kamruzzahan, A. S. M., Ebner, A., Wildling, L., Kienberger, F., Riener, C. K., Hahn, C. D., Pollheimer, P. D., Winklehner, P., Holzl, M., Lackner, B., Schorkl, D. M., Hinterdorfer, P., and Gruber, H. J. (2006) Antibody linking to atomic force microscope tips via disulfide bond formation, Bioconjugate Chemistry 17, 1473–1481.

    Article  PubMed  CAS  Google Scholar 

  44. Raab, A., Han, W., Badt, D., Smith-Gill, S. J., Lindsay, S. M., Schindler, H., and Hinterdorfer, P. (1999) Antibody recognition imaging by force microscopy, Nat Biotechnol 17, 901–905.

    Article  PubMed  CAS  Google Scholar 

  45. Riener, C. K., Stroh, C. M., Ebner, A., Klampfl, C., Gall, A. A., Romanin, C., Lyubchenko, Y. L., Hinterdorfer, P., and Gruber, H. J. (2003) Simple test system for single molecule recognition force microscopy, Analytica Chimica Acta 479, 59–75.

    Article  CAS  Google Scholar 

  46. Verbelen, C., Gruber, H. J., and Dufrene, Y. F. (2007) The NTA-HiS(6) bond is strong enough for AFM single-molecular recognition studies, in 1st International AFM BioMed Conference on Life Sciences and Medicine (AFM BioMed 2007), pp 490–494, John Wiley & Sons Ltd, Barcelona, SPAIN.

    Google Scholar 

  47. Dupres, V., Menozzi, F. D., Locht, C., Clare, B. H., Abbott, N. L., Cuenot, S., Bompard, C., Raze, D., and Dufrene, Y. F. (2005) Nanoscale mapping and functional analysis of individual adhesins on living ­bacteria, Nature Methods 2, 515–520.

    Article  PubMed  CAS  Google Scholar 

  48. Gad, M., Itoh, A., and Ikai, A. (1997) Mapping cell wall polysaccharides of living microbial cells using atomic force microscopy, Cell Biology International 21, 697–706.

    Article  PubMed  CAS  Google Scholar 

  49. Grandbois, M., Dettmann, W., Benoit, M., and Gaub, H. E. (2000) Affinity imaging of red blood cells using an atomic force microscope, Journal of Histochemistry & Cytochemistry 48, 719–724.

    Article  CAS  Google Scholar 

  50. Favis, B. D., Leonard, B. J., and Prudhomme, R. E. (1983) The interaction of a cationic silane coupling agent with mica, Journal of Applied Polymer Science 28, 1235–1244.

    Article  CAS  Google Scholar 

  51. Wang, H. D., Bash, R., Yodh, J. G., Hager, G. L., Lohr, D., and Lindsay, S. M. (2002) Glutaraldehyde modified mica: A new surface for atomic force microscopy of chromatin, Biophys. J. 83, 3619–3625.

    Article  PubMed  CAS  Google Scholar 

  52. Hutter, J. L., and Bechhoefer, J. (1993) Calibration of atomic-force microscope tips, Rev.Sci.Instr. 64, 1868–1873.

    Article  CAS  Google Scholar 

  53. Walters, D. A., Cleveland, J. P., Thomson, N. H., Hansma, P. K., Wendman, M. A., Gurley, G., and Elings, V. (1996) Short Cantilevers for Atomic-Force Microscopy, Rev.Sci.Instr. Vol 67, 3583–3590.

    Article  CAS  Google Scholar 

  54. Viani, M. B., Schaffer, T. E., Chand, A., Rief, M., Gaub, H. E., and Hansma, P. K. (1999) Small cantilevers for force spectroscopy of single molecules, J Appl Phys 86, 2258–2262.

    Article  CAS  Google Scholar 

  55. McManus, O. B., Blatz, A. L., and Magleby, K. L. (1987) Sampling, Binning, Fitting, And Plotting Durations Of Open-And-Shut Intervals From Single Channels, Biophys. J. 51, A48–A48.

    Google Scholar 

  56. Sigworth, F. J., and Sine, S. M. (1987) Data Transformations For Improved Display And Fitting Of Single-Channel Dwell Time Histograms, Biophys. J. 52, 1047–1054.

    Article  PubMed  CAS  Google Scholar 

  57. Pierres, A., Touchard, D., Benoliel, A. M., and Bongrand, P. (2002) Dissecting streptavidin-biotin interaction with a Laminar flow chamber, Biophys. J. 82, 3214–3223.

    Article  PubMed  CAS  Google Scholar 

  58. Bustamante, C., Marko, J. F., Siggia, E. D., and Smith, S. (1994) Entropic Elasticity of Lambda-Phage DNA, Science 265, 1599-1600.

    Article  PubMed  CAS  Google Scholar 

  59. Rief, M., Gautel, M., Oesterhelt, F., Fernandez, J. M., and Gaub, H. E. (1997) Reversible unfolding of individual titin immunoglobulin domains by AFM, Science 276, 1109–1112.

    Article  PubMed  CAS  Google Scholar 

  60. Hinterdorfer, P., Baumgartner, W., Gruber, H. J., Schilcher, K., and Schindler, H. (1996) Detection and localization of individual antibody-antigen recognition events by atomic force microscopy, Proceedings of the National Academy of Sciences of the United States of America 93, 3477–3481.

    Article  PubMed  CAS  Google Scholar 

  61. Rico, F., and Moy, V. T. (2007) Energy landscape roughness of the streptavidin-biotin interaction, Journal of Molecular Recognition 20, 495–501.

    Article  PubMed  CAS  Google Scholar 

  62. Tees, D. F. J., Waugh, R. E., and Hammer, D. A. (2001) A microcantilever device to assess the effect of force on the lifetime of selectin-carbohydrate bonds, Biophys.J. 80, 668–682.

    Article  PubMed  CAS  Google Scholar 

  63. Tees, D. F. J., Woodward, J. T., and Hammer, D. A. (2001) Reliability theory for receptor-ligand bond dissociation, J.Chem.Phys. 114, 7483–7496.

    Article  CAS  Google Scholar 

  64. Alcaraz, J. (2001) Micromechanics of Cultured Human Bronchial Epithelial Cells Measured with Atomic Force Microscopy, Universitat de Barcelona.

    Google Scholar 

  65. Janovjak, H. J., Struckmeier, J., and Muller, D. J. (2005) Hydrodynamic effects in fast AFM single-molecule force measurements, Eur Biophys J Biophy 34, 91–96.

    Article  CAS  Google Scholar 

  66. Kramers, H. A. (1940) Brownian motion in a field of force and the diffusion model of chemical reactions, Physica 7, 304.

    Article  Google Scholar 

  67. Hummer, G., and Szabo, A. (2005) Free energy surfaces from single-molecule force spectroscopy, Acc Chem Res 38, 504–513.

    Article  PubMed  CAS  Google Scholar 

  68. Friddle, R. W. (2008) Unified model of dynamic forced barrier crossing in single molecules, Physical Review Letters 100.

    Google Scholar 

  69. Evans, E. H., K; Kinoshita, K; Wong, WP. (2009) A new approach analysis of single-­molecule force measurements, in Handbook of single molecule biophysics (Hinterdorfer, P. v. O., A, Ed.), pp 571–589, Springer, New York.

    Google Scholar 

  70. Friddle, R. W., Podsiadlo, P., Artyukhin, A. B., and Noy, A. (2008) Near-equilibrium chemical force microscopy, Journal of Physical Chemistry C 112, 4986–4990.

    Article  CAS  Google Scholar 

  71. Zhang, Y., Sun, G. Y., Lu, S. Q., Li, N., and Long, M. A. (2008) Low Spring Constant Regulates P-Selectin-PSGL-1 Bond Rupture, Biophys. J. 95, 5439–5448.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Miller School of Medicine, University of Miami, Miami, FL, USA

    Vincent T. Moy

Authors
  1. Félix Rico
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Calvin Chu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Vincent T. Moy
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Vincent T. Moy .

Editor information

Editors and Affiliations

  1. School of Medicine, Department of Pharmacology, University of Milan, Via Vanvitelli 32, Milano, 20129, Italy

    Pier Carlo Braga

  2. , Robotics, Brain & Cognitive Sciences, Italian Institute of Technology, Via Morego 30, Genova, 16163, Italy

    Davide Ricci

Rights and permissions

Reprints and permissions

Copyright information

© 2011 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Rico, F., Chu, C., Moy, V.T. (2011). Force-Clamp Measurements of Receptor–Ligand Interactions. In: Braga, P., Ricci, D. (eds) Atomic Force Microscopy in Biomedical Research. Methods in Molecular Biology, vol 736. Humana Press. https://doi.org/10.1007/978-1-61779-105-5_20

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-105-5_20

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-104-8

  • Online ISBN: 978-1-61779-105-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation