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High-speed atomic force microscopy shows dynamic molecular processes in photoactivated bacteriorhodopsin


Dynamic changes in protein conformation in response to external stimuli are important in biological processes, but it has proved difficult to directly visualize such structural changes under physiological conditions1,2,3,4,5,6,7,8,9,10. Here, we show that high-speed atomic force microscopy7 can be used to visualize dynamic changes in stimulated proteins. High-resolution movies of a light-driven proton pump, bacteriorhodopsin11,12, reveal that, upon illumination, a cytoplasmic portion of each bacteriorhodopsin monomer is brought into contact with adjacent trimers. The bacteriorhodopsin–bacteriorhodopsin interaction in the transiently formed assembly engenders both positive and negative cooperative effects in the decay kinetics as the initial bacteriorhodopsin recovers and, as a consequence, the turnover rate of the photocycle is maintained constant, on average, irrespective of the light intensity. These results confirm that high-resolution visualization is a powerful approach for studying elaborate biomolecular processes under realistic conditions.

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Figure 1: High-speed AFM images of the cytoplasmic surface of D96N under dark or illuminated conditions.
Figure 2: Displacement of mass centre positions for WT at the cytoplasmic surface.
Figure 3: Cooperative effects on the decay kinetics in D96N.
Figure 4: Decay rate of activated D96N under different light intensities.


  1. 1

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

    CAS  Article  Google Scholar 

  2. 2

    Drake, B. et al. Imaging crystals, polymers and processes in water with the atomic force microscope. Science 243, 1586–1589 (1989).

    CAS  Article  Google Scholar 

  3. 3

    Butt, H. J., Downing, K. H. & Hansma, P. K. Imaging the membrane protein bacteriorhodopsin with the atomic force microscope. Biophys. J. 58, 1473–1480 (1990).

    CAS  Article  Google Scholar 

  4. 4

    Müller, D. J. et al. Atomic force microscopy of native purple membrane. Biochim. Biophys. Acta 1460, 27–38 (2000).

    Article  Google Scholar 

  5. 5

    Müller, D. J. AFM: a nanotool in membrane biology. Biochemistry 47, 7986–7998 (2008).

    Article  Google Scholar 

  6. 6

    Ando, T. et al. A high-speed atomic force microscope for studying biological macromolecules. Proc. Natl Acad. Sci. USA 98, 12468–12472 (2001).

    CAS  Article  Google Scholar 

  7. 7

    Ando, T., Uchihashi, T. & Fukuma, T. High-speed atomic force microscopy for nano-visualization of dynamic biomolecular processes. Prog. Sur. Sci. 83, 337–437 (2008).

    CAS  Article  Google Scholar 

  8. 8

    Hansma, P. K., Schitter, G., Fantner, G. E. & Prater, C. Applied physics. High-speed atomic force microscopy. Science 314, 601–602 (2006).

    CAS  Article  Google Scholar 

  9. 9

    Fantner, G. E. et al. Components for high speed atomic force microscopy. Ultramicroscopy 106, 881–887 (2006).

    CAS  Article  Google Scholar 

  10. 10

    Yamashita, H. et al. Tip–sample distance control using photothermal actuation of a small cantilever for high-speed atomic force microscopy. Rev. Sci. Instrum. 78, 083702 (2007).

    Article  Google Scholar 

  11. 11

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

    CAS  Article  Google Scholar 

  12. 12

    Lanyi, J. K. Bacteriorhodopsin. Annu. Rev. Physiol. 66, 665–688 (2004).

    CAS  Article  Google Scholar 

  13. 13

    Kimura, Y. et al. Surface of bacteriorhodopsin revealed by high-resolution electron crystallography. Nature 389, 206–211 (1997).

    CAS  Article  Google Scholar 

  14. 14

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

    CAS  Article  Google Scholar 

  15. 15

    Dencher, N. A., Dresselhaus, D., Zaccai, G. & Buldt, G. Structural changes in bacteriorhodopsin during proton translocation revealed by neutron diffraction. Proc. Natl Acad. Sci. USA 86, 7876–7879 (1989).

    CAS  Article  Google Scholar 

  16. 16

    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).

    CAS  Article  Google Scholar 

  17. 17

    Kamikubo, H. et al. Structure of the N intermediate of bacteriorhodopsin revealed by X-ray diffraction. Proc. Natl Acad. Sci. USA 93, 1386–1390 (1996).

    CAS  Article  Google Scholar 

  18. 18

    Thorgeirsson, T. E. et al. Transient channel-opening in bacteriorhodopsin: an EPR study. J. Mol. Biol. 273, 951–957 (1997).

    CAS  Article  Google Scholar 

  19. 19

    Brown, L. S., Needleman, R. & Lanyi, J. K. Conformational change of the E–F interhelical loop in the M photointermediate of bacteriorhodopsin. J. Mol. Biol. 317, 471–478 (2002).

    CAS  Article  Google Scholar 

  20. 20

    Shibata, M. & Kandori, H. FTIR studies of internal water molecules in the Schiff base region of bacteriorhodopsin. Biochemistry 44, 7406–7413 (2005).

    CAS  Article  Google Scholar 

  21. 21

    Sass, H. J. et al. Structural alterations for proton translocation in the M state of wild-type bacteriorhodopsin. Nature 406, 649–653 (2000).

    CAS  Article  Google Scholar 

  22. 22

    Luecke, H., Schobert, B., Richter, H. T., Cartailler, J. P. & Lanyi, J. K. Structural changes in bacteriorhodopsin during ion transport at 2 angstrom resolution. Science 286, 255–261 (1999).

    CAS  Article  Google Scholar 

  23. 23

    Subramaniam, S. & Henderson, R. Molecular mechanism of vectorial proton translocation by bacteriorhodopsin. Nature 406, 653–657 (2000).

    CAS  Article  Google Scholar 

  24. 24

    Vonck, J. Structure of the bacteriorhodopsin mutant F219 L N intermediate revealed by electron crystallography. EMBO J. 19, 2152–2160 (2000).

    CAS  Article  Google Scholar 

  25. 25

    Xiao, W., Brown, L. S., Needleman, R., Lanyi, J. K. & Shin, Y. K. Light-induced rotation of a transmembrane alpha-helix in bacteriorhodopsin. J. Mol. Biol. 304, 715–721 (2000).

    CAS  Article  Google Scholar 

  26. 26

    Otto, H. et al. Aspartic acid-96 is the internal proton donor in the reprotonation of the Schiff base of bacteriorhodopsin. Proc. Natl Acad. Sci. USA 86, 9228–9232 (1989).

    CAS  Article  Google Scholar 

  27. 27

    Heymann, J. B. et al. Charting the surfaces of the purple membrane. J. Struct. Biol. 128, 243–249 (1999).

    CAS  Article  Google Scholar 

  28. 28

    Korenstein, R., Hess, B. & Markus, M. Cooperativity in the photocycle of purple membrane of Halobacterium halobium with a mechanism of free energy transduction. FEBS Lett. 102, 155–161 (1979).

    CAS  Article  Google Scholar 

  29. 29

    Váró, G., Needleman, R. & Lanyi, J. K. Protein structural change at the cytoplasmic surface as the cause of cooperativity in the bacteriorhodopsin photocycle. Biophys. J. 70, 461–467 (1996).

    Article  Google Scholar 

  30. 30

    Oesterhelt, D. & Stoeckenius, W. Isolation of the cell membrane of Halobacterium halobium and its fractionation into red and purple membrane. Methods Enzymol. 31, 667–678 (1973).

    Article  Google Scholar 

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This work was supported by Japan Science and Technology Agency for Core Research for Evolutional Science and Technology (T.A.), Grants-in-Aids for Scientific Research from Japan Society for the Promotion of Science (JSPS) (no. 15101005; T.A.) and from the Ministry of Education, Culture, Sports, Science and Technology, Japan (no. 19042009, T.U.; no. 20108014, H.K.), and Research Fellowships of JSPS for Young Scientists (M.S. and H.Y.). The authors thank J.K. Lanyi, S.P. Balashov and L.S. Brown for comments on the draft.

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T.A., H.K. and M.S. conceived and designed the experiments. T.A. and T.U. developed the high-speed AFM instrument. T.U., M.S. and H.Y. performed the experiments. T.U. and M.S. analysed the data. T.A., T.U. and M.S. co-wrote the paper. All authors discussed the results and commented on the manuscript.

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Correspondence to Toshio Ando.

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The authors declare no competing financial interests.

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Shibata, M., Yamashita, H., Uchihashi, T. et al. High-speed atomic force microscopy shows dynamic molecular processes in photoactivated bacteriorhodopsin. Nature Nanotech 5, 208–212 (2010).

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