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

• Published in: Biochimica et biophysica acta. Biomembranes 2021-08, Vol.1863 (8), p.183637, Article 183637
• Main 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
• Format: Article
• Language: English
• Subjects: Chlorides - chemistry; Chlorides - metabolism; Chlorides - radiation effects; Fluorescence correlation spectroscopy; Halobacteriaceae - chemistry; Halobacteriaceae - genetics; Halorhodopsin; Halorhodopsins - chemistry; Halorhodopsins - genetics; Ion transport; Ion Transport - genetics; Kinetics; Light; Liposomes - chemistry; Liposomes - metabolism; Liposomes - radiation effects; Opsins; Stopped-flow
• ISSN: 0005-2736; 1879-2642
• DOI: 10.1016/j.bbamem.2021.183637

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.