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

Biochim Biophys Acta Biomembr. 2021 Aug 1;1863(8):183637. doi: 10.1016/j.bbamem.2021.183637. Epub 2021 Apr 27.

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%.

Keywords: Fluorescence correlation spectroscopy; Halorhodopsin; Ion transport; Opsins; Stopped-flow.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Chlorides / chemistry
  • Chlorides / metabolism*
  • Chlorides / radiation effects
  • Halobacteriaceae / chemistry
  • Halobacteriaceae / genetics
  • Halorhodopsins / chemistry*
  • Halorhodopsins / genetics
  • Ion Transport / genetics*
  • Kinetics
  • Light
  • Liposomes / chemistry*
  • Liposomes / metabolism
  • Liposomes / radiation effects

Substances

  • Chlorides
  • Halorhodopsins
  • Liposomes

Supplementary concepts

  • Natronomonas