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%.



Work Title Liposome-based measurement of light-driven chloride transport kinetics of halorhodopsin
Open Access
  1. Hasin Feroz
  2. Bryan Ferlez
  3. Hyeonji Oh
  4. Hossein Mohammadiarani
  5. Tingwei Ren
  6. Carol S. Baker
  7. John P. Gajewski
  8. Daniel J. Lugar
  9. Sandeep B. Gaudana
  10. Peter Butler
  11. Jonas Hühn
  12. Matthias Lamping
  13. Wolfgang J. Parak
  14. Michael R. Blatt
  15. Cheryl A. Kerfeld
  16. Nicholas Smirnoff
  17. Harish Vashisth
  18. John H. Golbeck
  19. Manish Kumar
License In Copyright (Rights Reserved)
Work Type Article
  1. Biochimica et Biophysica Acta - Biomembranes
Publication Date August 1, 2021
Publisher Identifier (DOI)
Deposited July 19, 2022




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Work History

Version 1

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