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

Academic Article


  • 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%.
  • Authors

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

    Publication Date

  • August 1, 2021
  • Keywords

  • Chlorides
  • Fluorescence correlation spectroscopy
  • Halobacteriaceae
  • Halorhodopsin
  • Halorhodopsins
  • Ion Transport
  • Ion transport
  • Kinetics
  • Light
  • Liposomes
  • Opsins
  • Stopped-flow
  • Digital Object Identifier (doi)

    Pubmed Id

  • 33930372
  • Start Page

  • 183637
  • Volume

  • 1863
  • Issue

  • 8