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

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%. [Display omitted] •Direct in-vitro measurement of the per protein transport rate of halorhodopsin (pHR)•Cl transport measured using fluorescent dye Cl− quenching in a stopped flow apparatus•Proteins per vesicle measured using fluorescence correlation spectroscopy (FCS)•Reconstituted pHR directionality determined using the pump inhibitor, HgCl2•Water-in-oil method of creating proteoliposomes leads to directional insertion of pHRs.