Origin of the Difference in Proton Transport Direction between Inward and Outward Proton-Pumping Rhodopsins

Abstract

Active transport is a vital and ubiquitous process in biological phenomena. Ion-pumping rhodopsins are light-driven active ion transporters that share a heptahelical transmembrane structural scaffold in which the all-trans retinal chromophore is covalently bonded through a Schiff base to a conserved lysine residue in the seventh transmembrane helix. Bacteriorhodopsin from Halobacterium salinarum was the first ion-pumping rhodopsin to be discovered and was identified as an outward proton-pumping rhodopsin. Since the discovery of bacteriorhodopsin in 1971, many more ion-pumping rhodopsins have been isolated from diverse microorganisms spanning three domains (bacteria, archaea, and eukaryotes) and giant viruses. In addition to proton-pumping rhodopsins, chloride ion- and sodium ion-pumping rhodopsins have also been discovered. Furthermore, diversity of ion-pumping rhodopsins was found in the direction of ion transport; i.e., rhodopsins that pump protons inward have recently been discovered. Very intriguingly, the inward proton-pumping rhodopsins share structural features and many conserved key residues with the outward proton-pumping rhodopsins. However, a central question remains unchanged despite the increasing variety: how and why do the ion-pumping rhodopsins undergo interlocking conformational changes that allow unidirectional ion transfer within proteins? In this regard, it is an effective strategy to compare the structures and their evolutions in the proton-pumping processes of both inward and outward proton-pumping rhodopsins because the comparison sheds light on key elements for the unidirectional proton transport. We elucidated the proton-pumping mechanism of the inward and outward proton-pumping rhodopsins by time-resolved resonance Raman spectroscopy, a powerful technique for tracking the structural evolutions of proteins at work that are otherwise inaccessible.