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Single molecule kinetics of bacteriorhodopsin by HS-AFM

Author

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  • Alma P. Perrino

    (Weill Cornell Medicine)

  • Atsushi Miyagi

    (Weill Cornell Medicine)

  • Simon Scheuring

    (Weill Cornell Medicine
    Weill Cornell Medicine)

Abstract

Bacteriorhodopsin is a seven-helix light-driven proton-pump that was structurally and functionally extensively studied. Despite a wealth of data, the single molecule kinetics of the reaction cycle remain unknown. Here, we use high-speed atomic force microscopy methods to characterize the single molecule kinetics of wild-type bR exposed to continuous light and short pulses. Monitoring bR conformational changes with millisecond temporal resolution, we determine that the cytoplasmic gate opens 2.9 ms after photon absorption, and stays open for proton capture for 13.2 ms. Surprisingly, a previously active protomer cannot be reactivated for another 37.6 ms, even under excess continuous light, giving a single molecule reaction cycle of ~20 s−1. The reaction cycle slows at low light where the closed state is prolonged, and at basic or acidic pH where the open state is extended.

Suggested Citation

  • Alma P. Perrino & Atsushi Miyagi & Simon Scheuring, 2021. "Single molecule kinetics of bacteriorhodopsin by HS-AFM," Nature Communications, Nature, vol. 12(1), pages 1-10, December.
    • Handle: RePEc:nat:natcom:v:12:y:2021:i:1:d:10.1038_s41467-021-27580-2
    DOI: 10.1038/s41467-021-27580-2
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    References listed on IDEAS

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    1. Gabriela Nass Kovacs & Jacques-Philippe Colletier & Marie Luise Grünbein & Yang Yang & Till Stensitzki & Alexander Batyuk & Sergio Carbajo & R. Bruce Doak & David Ehrenberg & Lutz Foucar & Raphael Gas, 2019. "Three-dimensional view of ultrafast dynamics in photoexcited bacteriorhodopsin," Nature Communications, Nature, vol. 10(1), pages 1-17, December.
    2. George R. Heath & Simon Scheuring, 2018. "High-speed AFM height spectroscopy reveals µs-dynamics of unlabeled biomolecules," Nature Communications, Nature, vol. 9(1), pages 1-11, December.
    3. Karl Edman & Peter Nollert & Antoine Royant & Hassan Belrhali & Eva Pebay-Peyroula & Janos Hajdu & Richard Neutze & Ehud M. Landau, 1999. "High-resolution X-ray structure of an early intermediate in the bacteriorhodopsin photocycle," Nature, Nature, vol. 401(6755), pages 822-826, October.
    4. Tina R. Matin & George R. Heath & Gerard H. M. Huysmans & Olga Boudker & Simon Scheuring, 2020. "Millisecond dynamics of an unlabeled amino acid transporter," Nature Communications, Nature, vol. 11(1), pages 1-11, December.
    5. George R. Heath & Ekaterina Kots & Janice L. Robertson & Shifra Lansky & George Khelashvili & Harel Weinstein & Simon Scheuring, 2021. "Localization atomic force microscopy," Nature, Nature, vol. 594(7863), pages 385-390, June.
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    Cited by:

    1. Yangang Pan & Jingyu Zhan & Yining Jiang & Di Xia & Simon Scheuring, 2023. "A concerted ATPase cycle of the protein transporter AAA-ATPase Bcs1," Nature Communications, Nature, vol. 14(1), pages 1-13, December.

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