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Determination of protein structural flexibility by microsecond force spectroscopy

Nature Nanotechnology volume 4, pages 514–517 (2009)Cite this article

Abstract

Proteins are dynamic molecular machines having structural flexibility that allows conformational changes1,2. Current methods for the determination of protein flexibility rely mainly on the measurement of thermal fluctuations and disorder in protein conformations3,4,5 and tend to be experimentally challenging. Moreover, they reflect atomic fluctuations on picosecond timescales, whereas the large conformational changes in proteins typically happen on micro- to millisecond timescales6,7. Here, we directly determine the flexibility of bacteriorhodopsin—a protein that uses the energy in light to move protons across cell membranes—at the microsecond timescale by monitoring force-induced deformations across the protein structure with a technique based on atomic force microscopy. In contrast to existing methods, the deformations we measure involve a collective response of protein residues and operate under physiologically relevant conditions with native proteins.

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Figure 1: Microsecond force spectroscopy in liquids.
Figure 2: High-resolution mapping of protein flexibility.
Figure 3: Flexibility analysis of bacteriorhodopsin with high-speed force–distance curves.

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References

  1. Falke, J. J. & Koshland, D. E. Global flexibility in a sensory receptor—a site-directed cross-linking approach. Science 237, 1596–1600 (1987).

    Article  CAS  Google Scholar 

  2. Frauenfelder, H., Sligar, S. G. & Wolynes, P. G. The energy landscapes and motions of proteins. Science 254, 1598–1603 (1991).

    Article  CAS  Google Scholar 

  3. Frauenfelder, H., Petsko, G. A. & Tsernoglou, D. Temperature-dependent X-ray-diffraction as a probe of protein structural dynamics. Nature 280, 558–563 (1979).

    Article  CAS  Google Scholar 

  4. Mittermaier, A. & Kay, L. E. New tools provide new insights in NMR studies of protein dynamics. Science 312, 224–228 (2006).

    Article  CAS  Google Scholar 

  5. Zaccai, G. How soft is a protein? A protein dynamics force constant measured by neutron scattering. Science 288, 1604–1607 (2000).

    Article  CAS  Google Scholar 

  6. Subramaniam, S., Gerstein, M., Oesterhelt, D. & Henderson, R. Electron-diffraction analysis of structural changes in the photocycle of bacteriorhodopsin. EMBO J. 12, 1–8 (1993).

    Article  CAS  Google Scholar 

  7. Rousso, I. et al. Microsecond atomic force sensing of protein conformational dynamics: implications for the primary light-induced events in bacteriorhodopsin. Proc. Natl Acad. Sci. USA 94, 7937–7941 (1997).

    Article  CAS  Google Scholar 

  8. Dodson, G. & Verma, C. S. Protein flexibility: its role in structure and mechanism revealed by molecular simulations. Cell. Mol. Life Sci. 63, 207–219 (2006).

    Article  CAS  Google Scholar 

  9. Clausen-Schaumann, H., Seitz, M., Krautbauer, R. & Gaub, H. E. Force spectroscopy with single bio-molecules. Curr. Opin. Chem. Biol. 4, 524–530 (2000).

    Article  CAS  Google Scholar 

  10. Sulchek, T. A. et al. Dynamic force spectroscopy of parallel individual Mucin1-antibody bonds. Proc. Natl Acad. Sci. USA 102, 16638–16643 (2005).

    Article  CAS  Google Scholar 

  11. Dietz, H., Berkemeier, F., Bertz, M. & Rief, M. Anisotropic deformation response of single protein molecules. Proc. Natl Acad. Sci. USA 103, 12724–12728 (2006).

    Article  CAS  Google Scholar 

  12. Knowles, T. P. et al. Role of intermolecular forces in defining material properties of protein nanofibrils. Science 318, 1900–1903 (2007).

    Article  CAS  Google Scholar 

  13. Sahin, O., Magonov, S., Su, C., Quate, C. F. & Solgaard, O. An atomic force microscope tip designed to measure time-varying nanomechanical forces. Nature Nanotech. 2, 507–514 (2007).

    Article  Google Scholar 

  14. Martinez, N. F. et al. Bimodal atomic force microscopy imaging of isolated antibodies in air and liquids. Nanotechnology 19, 384011 (2008).

    Article  CAS  Google Scholar 

  15. Preiner, J., Tang, J. L., Pastushenko, V. & Hinterdorfer, P. Higher harmonic atomic force microscopy: Imaging of biological membranes in liquid. Phys. Rev. Lett. 99, 046102 (2007).

    Article  Google Scholar 

  16. Legleiter, J., Park, M., Cusick, B. & Kowalewski, T. Scanning probe acceleration microscopy (SPAM) in fluids: Mapping mechanical properties of surfaces at the nanoscale. Proc. Natl Acad. Sci. USA 103, 4813–4818 (2006).

    Article  CAS  Google Scholar 

  17. Haupts, U., Tittor, J. & Oesterhelt, D. Closing in on bacteriorhodopsin: progress in understanding the molecule. Annu. Rev. Biophys. Biomol. Struct. 28, 367–399 (1999).

    Article  CAS  Google Scholar 

  18. Luecke, H., Schobert, B., Richter, H. T., Cartailler, J. P. & Lanyi, J. K. Structure of bacteriorhodopsin at 1.55 angstrom resolution. J. Mol. Biol. 291, 899–911 (1999).

    Article  CAS  Google Scholar 

  19. Muller, D. J. & Engel, A. The height of biomolecules measured with the atomic force microscope depends on electrostatic interactions. Biophys. J. 73, 1633–1644 (1997).

    Article  CAS  Google Scholar 

  20. Zhong, S. et al. Different interactions between the two sides of purple membrane with atomic force microscope tip. Langmuir 23, 4486–4493 (2007).

    Article  CAS  Google Scholar 

  21. Sahin, O. Harnessing bifurcations in tapping-mode atomic force microscopy to calibrate time-varying tip–sample force measurements. Rev. Sci. Instrum. 78, 103707 (2007).

    Article  Google Scholar 

  22. Parlak, Z. & Degertekin, F. L. Contact stiffness of finite size subsurface defects for atomic force microscopy: three-dimensional finite element modeling and experimental verification. J. Appl. Phys. 103, 114910 (2008).

    Article  Google Scholar 

  23. Stark, M., Moller, C., Muller, D. J. & Guckenberger, R. From images to interactions: high-resolution phase imaging in tapping-mode atomic force microscopy. Biophys. J. 80, 3009–3018 (2001).

    Article  CAS  Google Scholar 

  24. Alexiev, U., Marti, T., Heyn, M. P., Khorana, H. G. & Scherrer, P. Surface-charge of bacteriorhodopsin detected with covalently bound pH indicators at selected extracellular and cytoplasmic sites. Biochemistry 33, 298–306 (1994).

    Article  CAS  Google Scholar 

  25. Engel, A. & Muller, D. J. Observing single biomolecules at work with the atomic force microscope. Nature Struct. Biol. 7, 715–718 (2000).

    Article  CAS  Google Scholar 

  26. Muller, D. J., Fotiadis, D. & Engel, A. Mapping flexible protein domains at subnanometer resolution with the atomic force microscope. FEBS Lett. 430, 105–111 (1998).

    Article  CAS  Google Scholar 

  27. Muller, D. J. & Engel, A. Atomic force microscopy and spectroscopy of native membrane proteins. Nature Protoc. 2, 2191–2197 (2007).

    Article  Google Scholar 

  28. Patrick, D. L., Cee, V. J. & Beebe, T. P. Molecule corrals for studies of monolayer organic films. Science 265, 231–234 (1994).

    Article  CAS  Google Scholar 

  29. Israelachvili, J. Intermolecular and Surface Forces (Academic Press, 2003).

    Google Scholar 

  30. Johnson, K. L. Contact Mechanics (Cambridge Univ. Press, 2003).

    Google Scholar 

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Acknowledgements

This work was supported by the Rowland Junior Fellows program.

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Authors and Affiliations

  1. Rowland Institute at Harvard, Harvard University, Cambridge, 02142, Massachusetts, USA

    Mingdong Dong, Sudhir Husale & Ozgur Sahin

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  1. Mingdong Dong
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  2. Sudhir Husale
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  3. Ozgur Sahin
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Correspondence to Ozgur Sahin.

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Dong, M., Husale, S. & Sahin, O. Determination of protein structural flexibility by microsecond force spectroscopy. Nature Nanotech 4, 514–517 (2009). https://doi.org/10.1038/nnano.2009.156

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