Liposome-based measurement of light-driven chloride transport kinetics of halorhodopsin

Hasin Feroz, Bryan Ferlez, Hyeonji Oh, Hossein Mohammadiarani, Tingwei Ren, Carol S. Baker, John P. Gajewski, Daniel J. Lugar, Sandeep B. Gaudana, Peter Butler, Jonas Hühn, Matthias Lamping, Wolfgang J. Parak, Michael R. Blatt, Cheryl A. Kerfeld, Nicholas Smirnoff, Harish Vashisth, John H. Golbeck, Manish Kumar

Research output: Contribution to journalArticlepeer-review

Abstract

We report a simple and direct fluorimetric vesicle-based method for measuring the transport rate of the light-driven ions pumps as specifically applied to the chloride pump, halorhodopsin, from Natronomonas pharaonis (pHR). Previous measurements were cell-based and methods to determine average single channel permeability challenging. We used a water-in-oil emulsion method for directional pHR reconstitution into two different types of vesicles: lipid vesicles and asymmetric lipid-block copolymer vesicles. We then used stopped-flow experiments combined with fluorescence correlation spectroscopy to determine per protein Cl- transport rates. We obtained a Cl transport rate of 442 (±17.7) Cl/protein/s in egg phosphatidyl choline (PC) lipid vesicles and 413 (±26) Cl/protein/s in hybrid block copolymer/lipid (BCP/PC) vesicles with polybutadine-polyethylene oxide (PB12PEO8) on the outer leaflet and PC in the inner leaflet at a photon flux of 1450 photons/protein/s. Normalizing to a per photon basis, this corresponds to 0.30 (±0.07) Cl/photon and 0.28 (±0.04) Cl/photon for pure PC and BCP/PC hybrid vesicles respectively, both of which are in agreement with recently reported turnover of ~500 Cl/protein/s from flash photolysis experiments and with voltage-clamp measurements of 0.35 (±0.16) Cl/photon in pHR-expressing oocytes as well as with a pHR quantum efficiency of ~30%.

Original languageEnglish (US)
Article number183637
JournalBiochimica et Biophysica Acta - Biomembranes
Volume1863
Issue number8
DOIs
StatePublished - Aug 1 2021

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Cell Biology

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