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Single Molecule Studies of Protein Folding Using Atomic Force Microscopy

  • Protocol
Book cover Protein Folding Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 350))

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Abstract

Atomic force microscopy (AFM) offers new insights into the ability of proteins to resist mechanical force. The technique has been opened up by the availability of easy-to-use instruments that are commercially available, so that the technique no longer relies on the need to build instruments in the lab. Indeed it may become common for AFM instruments to sit beside stopped-flow apparatus in protein folding laboratories. In this chapter, we describe the instrument set-up, the preparation of suitable protein substrate, and the collection of data. Data selection and analysis are more complex than for conventional stopped-flow ensemble studies, but offer new insights into the function of proteins in vivo.

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References

  1. Binnig, G., Quate, C. F., and Gerber, C. (1986) Atomic force microscope. Phys. Rev. Lett. 56, 930–933.

    Article  PubMed  Google Scholar 

  2. Lee, G. U., Chrisey, L. A., and Colton, R. J. (1994) Direct measurement of the forces between complementary strands of DNA. Science 266, 771–773.

    Article  CAS  PubMed  Google Scholar 

  3. Marszalek, P. E., Li, H., and Fernandez, J. M. (2001) Fingerprinting polysaccharides with single-molecule atomic force microscopy. Nature Biotechnol. 19, 258–262.

    Article  CAS  Google Scholar 

  4. 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  CAS  PubMed  Google Scholar 

  5. Halliday, N. L. and Tomasek, J. J. (1995) Mechanical properties of the extracellular matrix influence fibronectin fibril assembly in vitro. Expt. Cell Res. 217, 109–117.

    Article  CAS  Google Scholar 

  6. Hynes, R. O. (1999) The dynamic dialogue between cells and matrices: implications of fibronectin’s elasticity. Proc. Natl Acad. Sci. USA 96, 2588–2590.

    Article  CAS  PubMed  Google Scholar 

  7. Sharma, A., Askari, J. A., Humphries, M. J., Jones, E. Y., and Stuart, D. I. (1999) Crystal structure of a heparin-and integrin-binding segment of human fibronectin. EMBO J. 18, 1468–1479.

    Article  CAS  PubMed  Google Scholar 

  8. Sechler, J. L., Rao, H., Cumiskey, A. M., et al. (2001) A novel fibronectin binding site required for fibronectin fibril growth during matrix assembly. J. Cell Biol. 154, 1081–1088.

    Article  CAS  PubMed  Google Scholar 

  9. Maruyama, K. (1997) Connectin/titin, giant elastic protein of muscle. FASEB J. 11, 341–345.

    CAS  PubMed  Google Scholar 

  10. Labeit, S. and Kolmerer, B. (1995) Titins: giant proteins in charge of muscle ultrastructure and elasticity. Science 270, 293–296.

    Article  CAS  PubMed  Google Scholar 

  11. Wang, K., McCarter, R., Wright, J., Beverly, J., and Ramirez-Mitchell, R. (1991) Regulation of skeletal muscle stiffness and elasticity by titin isoforms: a test of the segmental extension model of resting tension. Proc. Natl Acad. Sci. USA 88, 7101–7105.

    Article  CAS  PubMed  Google Scholar 

  12. Wang, K., McClure, J., and Tu, A. (1979) Titin: major myofibrillar components of striated muscle. Proc. Natl Acad. Sci. USA 76, 3698–3702.

    Article  CAS  PubMed  Google Scholar 

  13. Carrion-Vazquez, M., Li, H., Lu, H., Marszalek, P. E., Oberhauser, A. F., and Fernandez, J. M. (2003). The mechanical stability of ubiquitin is linkage dependent. Nature Struct. Biol. 10, 738–743.

    Article  CAS  PubMed  Google Scholar 

  14. Brockwell, D. J., Paci, E., Zinober, R. C., et al. (2003) Pulling geometry defines the mechanical resistance of a beta-sheet protein. Nature Struct. Biol. 10, 731–737.

    Article  CAS  PubMed  Google Scholar 

  15. Li, L., Huang, H. H., Badilla, C. L., and Fernandez, J. M. (2005) Mechanical unfolding intermediates observed by single-molecule force spectroscopy in a fibronectin type III module. J. Mol. Biol. 345, 817–826.

    Article  CAS  PubMed  Google Scholar 

  16. Best, R. B., Fowler, S. B., Herrera, J. L., Steward, A., Paci, E., and Clarke, J. (2003) Mechanical unfolding of a titin Ig domain: structure of transition state revealed by combining atomic force microscopy, protein engineering and molecular dynamics simulations. J. Mol. Biol. 330, 867–877.

    Article  CAS  PubMed  Google Scholar 

  17. Fowler, S. B., Best, R. B., Toca Herrera, J. L., et al. (2002) Mechanical unfolding of a titin Ig domain: structure of unfolding intermediate revealed by combining AFM, molecular dynamics simulations, NMR and protein engineering. J. Mol. Biol. 322, 841–849.

    Google Scholar 

  18. Marszalek, P. E., Lu, H., Li, H., et al. (1999) Mechanical unfolding intermediates in titin modules. Nature 402, 100–103.

    Article  CAS  PubMed  Google Scholar 

  19. Fernandez, J. M. and Li, H. (2004) Force-clamp spectroscopy monitors the folding trajectory of a single protein. Science 303, 1674–1678.

    Article  CAS  PubMed  Google Scholar 

  20. Oberhauser, A. F., Marszalek, P. E., Carrion-Vazquez, M., and Fernandez, J. M. (1999) Single protein misfolding events captured by atomic force microscopy. Nature Struct. Biol. 6, 1025–1028.

    Article  CAS  PubMed  Google Scholar 

  21. Steward, A., Toca-Herrera, J. L., and Clarke, J. (2002) Versatile cloning system for construction of multimeric proteins for use in atomic force microscopy. Protein Sci. 11, 2179–2183.

    Article  CAS  PubMed  Google Scholar 

  22. Fersht, A. R., Matouschek, A., and Serrano, L. (1992) The folding of an enzyme. I. Theory of protein engineering analysis of stability and pathway of protein folding. J. Mol. Biol. 224, 771–782.

    Article  CAS  PubMed  Google Scholar 

  23. Pain, R. H. (2000) Mechanisms of Protein Folding, Oxford University Press.

    Google Scholar 

  24. Bell, G. I. (1978) Models for the specific adhesion of cells to cells. Science 200, 618–627.

    Article  CAS  PubMed  Google Scholar 

  25. Bustamante, C., Marko, J. F., Siggia, E. D., and Smith, S. (1994) Entropic elasticity of lambda-phage DNA. Science 265, 1599, 1600.

    Article  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  27. Gamsjaeger, R., Wimmer, B., Kahr, H., et al. (2004) Oriented binding of the His(6)-tagged carboxyl-tail of the L-type Ca2+ channel alpha(1)-subunit to a new NTA-functionalized self-assembled monolayer. Langmuir 20, 5885–5893.

    Article  CAS  PubMed  Google Scholar 

  28. 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. USA 93, 3477–3481.

    Article  CAS  PubMed  Google Scholar 

  29. Cleveland, J. P., Manne, S., Bocek, D., and Hansma, P. K. (1993) A nondestructive method for determining the spring constant of cantilevers for scanning force microscopy. Rev. Sci. Instrum. 64, 403–405.

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  31. Proksch, R. (2004) Nondestructive added mass spring calibration with the MFP-3D. Technical Note of Asylum Research. http://www.AsylumResearch.com. Last accessed March 17, 2006.

  32. Law, R., Liao, G., Harper, S., Yang, G., Speicher, D. W., and Discher, D. E. (2003) Pathway shifts and thermal softening in temperature-coupled forced unfolding of spectrin domains. Biophys. J. 85, 3286–3293.

    Article  CAS  PubMed  Google Scholar 

  33. Oberhauser, A. F., Marszalek, P. E., Erickson, H. P., and Fernandez, J. M. (1998) The molecular elasticity of the extracellular matrix protein tenascin. Nature 393, 181–185.

    Article  CAS  PubMed  Google Scholar 

  34. Carrion-Vazquez, M., Oberhauser, A. F., Fowler, S. B., et al. (1999) Mechanical and chemical unfolding of a single protein: a comparison. Proc. Natl Acad. Sci. USA 96, 3694–3699.

    Article  CAS  PubMed  Google Scholar 

  35. Politou, A. S., Gautel, M., Joseph, C., and Pastore, A. (1994) Immunoglobulin-type domains of titin are stabilized by amino-terminal extension. FEBS Lett. 352, 27–31.

    Article  CAS  PubMed  Google Scholar 

  36. Pfuhl, M., Improta, S., Politou, A. S., and Pastore, A. (1997) When a module is also a domain: the role of the N terminus in the stability and the dynamics of immunoglobulin domains from titin. J. Mol. Biol. 265, 242–256.

    Article  CAS  PubMed  Google Scholar 

  37. Hamill, S. J., Meekhof, A. E., and Clarke, J. (1998) The effect of boundary selection on the stability and folding of the third fibronectin type III domain from human tenascin. Biochemistry 37, 8071–8079.

    Article  CAS  PubMed  Google Scholar 

  38. Miroux, B. and Walker, J. E. (1996) Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J. Mol. Biol. 260, 289–298.

    Article  CAS  PubMed  Google Scholar 

  39. Best, R. B., Li, B., Steward, A., Daggett, V., and Clarke, J. (2001) Can nonmechanical proteins withstand force? Stretching barnase by atomic force microscopy and molecular dynamics simulation. Biophys. J. 81, 2344–2356.

    Article  CAS  PubMed  Google Scholar 

  40. Forman, J. R., Qamar, S., Paci, E., Sandford, R. N., and Clarke, J. (2005) The remarkable mechanical strength of polycystin-1 supports a direct role in mechanotransduction. J. Mol. Biol. 349, 861–871.

    Article  CAS  PubMed  Google Scholar 

  41. Rounsevell, R. W. S., Steward, A., and Clarke, J. (2005) Biophysical investigations of engineered polyproteins: implications for force data. Biophys. J. 88, 2022–2029.

    Article  CAS  PubMed  Google Scholar 

  42. Evans, E. and Williams, P. M. (2002) Dynamic Force Spectroscopy I: Single Bonds in Les Houches-Ecole d’Ete de Physique Theorique. Springer and Verlag, Germany.

    Google Scholar 

  43. Rief, M., Fernandez, J. M., and Gaub, H. E. (1998) Elastically coupled two-level systems as a model for biopolymer extensibility. Phys. Rev. Lett. 81, 4764–4767.

    Article  CAS  Google Scholar 

  44. Clarke, J. and Williams, P. M. (2005) Unfolding induced by mechanical force. In: Protein Folding Handbook Part 1 (Kiefhaber, T. and Buchner, J., ed.), Wiley-VCH Verlag GmbH and Co., Weinheim, Germany, pp. 1111–1142.

    Chapter  Google Scholar 

  45. Best, R. B., Brockwell, D. J., Toca-Herrera, J. L., et al. (2003) Force mode atomic force microscopy as a tool for protein folding studies. Anal. Chim. Acta, 479, 87–105.

    Article  CAS  Google Scholar 

  46. Ng, S. P., Rounsevell, R. W., Steward, A., et al. (2005) Mechanical unfolding of TNfn3: the unfolding pathway of a fnIII domain probed by protein engineering, AFM and MD simulation. J. Mol. Biol. 350, 776–789.

    Article  CAS  PubMed  Google Scholar 

  47. Best, R. B., Fowler, S. B., Toca-Herrera, J. L., and Clarke, J. (2002) A simple method for probing the mechanical unfolding pathway of proteins in detail. Proc. Natl Acad. Sci. USA 99, 12,143–12,148.

    Article  CAS  PubMed  Google Scholar 

  48. Zinober, R. C., Brockwell, D. J., Beddard, G. S., et al. (2002) Mechanically unfolding proteins: the effect of unfolding history and the supramolecular scaffold. Protein Sci. 11, 2759–2765.

    Article  CAS  PubMed  Google Scholar 

  49. Evans, E. (2001) Probing the relation between force—lifetime—and chemistry in single molecular bonds. Annu. Rev. Biophys. Biomol. Struct. 30, 105–128.

    Article  CAS  PubMed  Google Scholar 

  50. Schwaiger, I., Kardinal, A., Schleicher, M., Noegel, A. A., and Rief, M. (2004) A mechanical unfolding intermediate in an actin-crosslinking protein. Nature Struct. Mol. Biol. 11, 81–85.

    Article  CAS  Google Scholar 

  51. Williams, P. M., Fowler, S. B., Best, R. B., et al. (2003) Hidden complexity in the mechanical properties of titin. Nature 422, 446–449.

    Article  CAS  PubMed  Google Scholar 

  52. Rounsevell, R., Forman, J. R., and Clarke, J. (2004) Atomic force microscopy: mechanical unfolding of proteins. Methods 34, 100–111.

    Article  CAS  PubMed  Google Scholar 

  53. Li, H., Oberhauser, A. F., Fowler, S. B., Clarke, J., and Fernandez, J. M. (2000) Atomic force microscopy reveals the mechanical design of a modular protein. Proc. Natl Acad. Sci. USA 97, 6527–6531.

    Article  CAS  PubMed  Google Scholar 

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Author information

Authors and Affiliations

  1. Department of Chemistry, University of Cambridge, Medical Research Council Center for Protein Engineering, Cambridge, UK

    Sean P. Ng, Lucy G. Randles & Jane Clarke

Authors
  1. Sean P. Ng
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  2. Lucy G. Randles
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  3. Jane Clarke
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Editor information

Editors and Affiliations

  1. Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, MD

    Yawen Bai

  2. Center for Cancer Research Nanobiology Program SAIC, National Cancer Institute-Frederick, Frederick, MD

    Ruth Nussinov

  3. Department of Human Genetics, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel

    Ruth Nussinov

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© 2007 Humana Press Inc.

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Ng, S.P., Randles, L.G., Clarke, J. (2007). Single Molecule Studies of Protein Folding Using Atomic Force Microscopy. In: Bai, Y., Nussinov, R. (eds) Protein Folding Protocols. Methods in Molecular Biology™, vol 350. Humana Press. https://doi.org/10.1385/1-59745-189-4:139

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  • DOI: https://doi.org/10.1385/1-59745-189-4:139

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-622-1

  • Online ISBN: 978-1-59745-189-5

  • eBook Packages: Springer Protocols

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