Light-Driven Chloride Transport Kinetics of Halorhodopsin

Hasin Feroz, Bryan Ferlez, Cecile Lefoulon, Tingwei Ren, Carol S. Baker, John P. Gajewski, Daniel J. Lugar, Sandeep B. Gaudana, Peter J. Butler, Jonas Hühn, Matthias Lamping, Wolfgang J. Parak, Julian M. Hibberd, Cheryl A. Kerfeld, Nicholas Smirnoff, Michael R. Blatt, John H. Golbeck, Manish Kumar

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Abstract

Despite growing interest in light-driven ion pumps for use in optogenetics, current estimates of their transport rates span two orders of magnitude due to challenges in measuring slow transport processes and determining protein concentration and/or orientation in membranes in vitro. In this study, we report, to our knowledge, the first direct quantitative measurement of light-driven Cl transport rates of the anion pump halorohodopsin from Natronomonas pharaonis (NpHR). We used light-interfaced voltage clamp measurements on NpHR-expressing oocytes to obtain a transport rate of 219 (± 98) Cl/protein/s for a photon flux of 630 photons/protein/s. The measurement is consistent with the literature-reported quantum efficiency of ∼30% for NpHR, i.e., 0.3 isomerizations per photon absorbed. To reconcile our measurements with an earlier-reported 20 ms rate-limiting step, or 35 turnovers/protein/s, we conducted, to our knowledge, novel consecutive single-turnover flash experiments that demonstrate that under continuous illumination, NpHR bypasses this step in the photocycle.

Original languageEnglish (US)
Pages (from-to)353-360
Number of pages8
JournalBiophysical journal
Volume115
Issue number2
DOIs
StatePublished - Jul 17 2018

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All Science Journal Classification (ASJC) codes

  • Biophysics

Cite this

Feroz, H., Ferlez, B., Lefoulon, C., Ren, T., Baker, C. S., Gajewski, J. P., Lugar, D. J., Gaudana, S. B., Butler, P. J., Hühn, J., Lamping, M., Parak, W. J., Hibberd, J. M., Kerfeld, C. A., Smirnoff, N., Blatt, M. R., Golbeck, J. H., & Kumar, M. (2018). Light-Driven Chloride Transport Kinetics of Halorhodopsin. Biophysical journal, 115(2), 353-360. https://doi.org/10.1016/j.bpj.2018.06.009