Protein-Bound Water as the Determinant of Asymmetric Functional Conversion between Light-Driven Proton and Chloride Pumps

Abstract

Bacteriorhodopsin (BR) and halorhodopsin (HR) are light-driven outward proton and inward chloride pumps, respectively. They have similar protein architecture, being composed of seven-transmembrane helices that bind an all-trans-retinal. BR can be converted into a chloride pump by a single amino acid replacement at position 85, suggesting that BR and HR share a common transport mechanism, and the ionic specificity is determined by the amino acid at that position. However, HR cannot be converted into a proton pump by the corresponding reverse mutation. Here we mutated 6 and 10 amino acids of HR into BR-like, whereas such multiple HR mutants never pump protons. Light-induced Fourier transform infrared spectroscopy revealed that hydrogen bonds of the retinal Schiff base and water are both strong for BR and both weak for HR. Multiple HR mutants exhibit strong hydrogen bonds of the Schiff base, but the hydrogen bond of water is still weak. We concluded that the cause of nonfunctional conversion of HR is the lack of strongly hydrogen-bonded water, the functional determinant of the proton pump.

Halorhodopsin pumps Cl–and bacteriorhodopsin pumps protons by a common mechanism that uses conserved electrostatic interactions

“…They also require control of the accessibility to intracellular and extracellular compartments through the reaction cycle, which has not been considered in the work reported here. In addition, proton pumping requires long-range hydrogen bonded networks to transfer the H + , which are not needed for ion transfer (31). Based on the finding reported here, we would suggest a doublemutant BR-D85T/E204T may be a more efficient Cl -pump than the single-mutant D85T, as it should bind chloride more effectively.…”

“…This has proved to be more difficult than turning BR to a chloride pump (31). Both proton and Cl -pumping require changes in affinity, which has been explored here.…”

“…A single BR-Asp85 to Ser (29), Thr (29), or Asn (30) substitution converts BR into a chloride pump. On the other hand, it has proved harder to mutate HR to a proton pump (31). An HR with six BR amino acids added along the transfer pathway does not pump protons.…”

A light-driven sodium ion pump in marine bacteria

“…E. coli expressing rhodopsins was prepared in manner previously described 52,53 . The cells were collected by centrifugation (4,800g, 3 min), washed three times and resuspended in the solvent for measurement.…”

“…KR1 and KR2 having six histidines at the C terminus were expressed in E. coli C41(DE3) strain. The protein was purified via Co-affinity column (TALON, Qiagen) chromatography in manner previously described 52,53 and solubilized in 0.1% n-dodecyl-b-D-maltoside (DDM).…”