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

doi:10.1016/j.bpj.2018.06.009

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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7 Scopus citations

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 language	English (US)
Pages (from-to)	353-360
Number of pages	8
Journal	Biophysical journal
Volume	115
Issue number	2
DOIs	https://doi.org/10.1016/j.bpj.2018.06.009
State	Published - Jul 17 2018

All Science Journal Classification (ASJC) codes

Biophysics

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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

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title = "Light-Driven Chloride Transport Kinetics of Halorhodopsin",

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.",

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

note = "Funding Information: We thank Shared Fermentation Facility (The Huck Institutes of The Life Sciences, Pennsylvania State University) for support with the protein purification and National Science Foundation for providing funding (PIs: J.H.G. and M.K.) through grant MCB-1359634 and (PIs: C.A.K.) through grant MCB-1359636 . Parts of the project were supported by the German Research Foundation (DFG grant 794/21-1 to W.J.P.) and by the National Science Foundation grant CBET-1552571 to M.K.. M.B. acknowledges support from grants BB/M01133X/1 , BB/M001601/1 , and BB/L019025/1 from the UK Biotechnology and Biological Sciences Research Council . Publisher Copyright: {\textcopyright} 2018 Biophysical Society",

year = "2018",

month = jul,

day = "17",

doi = "10.1016/j.bpj.2018.06.009",

language = "English (US)",

volume = "115",

pages = "353--360",

journal = "Biophysical Journal",

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publisher = "Biophysical Society",

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TY - JOUR

T1 - Light-Driven Chloride Transport Kinetics of Halorhodopsin

AU - Feroz, Hasin

AU - Ferlez, Bryan

AU - Lefoulon, Cecile

AU - Ren, Tingwei

AU - Baker, Carol S.

AU - Gajewski, John P.

AU - Lugar, Daniel J.

AU - Gaudana, Sandeep B.

AU - Butler, Peter J.

AU - Hühn, Jonas

AU - Lamping, Matthias

AU - Parak, Wolfgang J.

AU - Hibberd, Julian M.

AU - Kerfeld, Cheryl A.

AU - Smirnoff, Nicholas

AU - Blatt, Michael R.

AU - Golbeck, John H.

AU - Kumar, Manish

N1 - Funding Information: We thank Shared Fermentation Facility (The Huck Institutes of The Life Sciences, Pennsylvania State University) for support with the protein purification and National Science Foundation for providing funding (PIs: J.H.G. and M.K.) through grant MCB-1359634 and (PIs: C.A.K.) through grant MCB-1359636 . Parts of the project were supported by the German Research Foundation (DFG grant 794/21-1 to W.J.P.) and by the National Science Foundation grant CBET-1552571 to M.K.. M.B. acknowledges support from grants BB/M01133X/1 , BB/M001601/1 , and BB/L019025/1 from the UK Biotechnology and Biological Sciences Research Council . Publisher Copyright: © 2018 Biophysical Society

PY - 2018/7/17

Y1 - 2018/7/17

N2 - 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.

AB - 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.

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U2 - 10.1016/j.bpj.2018.06.009

DO - 10.1016/j.bpj.2018.06.009

M3 - Article

C2 - 30021110

AN - SCOPUS:85049338671

SN - 0006-3495

VL - 115

SP - 353

EP - 360

JO - Biophysical Journal

JF - Biophysical Journal

IS - 2

ER -

Light-Driven Chloride Transport Kinetics of Halorhodopsin

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