Thermal Equilibrium and Energy Conversion of a New Photoreceptor with Two Chromophores as Studied by In Situ Spectroscopy

Abstract

Archaerhodopsin-4 (aR4), an unknown structured membrane protein from Halobacterium species XZ515 found in a salt lake in China, functions as a proton pump similar to that of bacteriorhodopsin (bR). It has a seven-transmembrane topology and a protonated Schiff base formed by the retinal chromophore with the lysine 217 on the helix G through a covalent bond linkage. Absorption of a photon causes photoisomerization of the retinal chromophore from the all-trans to the 13-cis, 15-anti configuration and triggers a series of structural rearrangements of the protein that initiates a vectorial translocation of a proton out of the cell. aR4 has not only a retinal as the premier chromophore, but also a bacterioruberin as the second chromophore. Furthermore, aR4 has an opposite temporal order of proton uptake and release at neutral pH as compared with bR. In order to elucidate how the retinal cis-trans thermal equilibrium is maintained and modulated by the second chromophore, and further affect the proton uptake and release order, the photocycle kinetics, and the energy conversion efficiency in the claret membrane, in situ 2D solid-state NMR of the specifically labelled receptors in the claret membranes, reinforced with many single point mutation analyses and function assays were carried out. Hydrogen bonding and hydrophobic interactions were identified as the mechanistic origin of multiple outcomes, including direct electromechanical coupling to the dynamics of conformational changes within the receptor, direct monomer interactions, and energy conversion efficiency in native membranes. These new insights may be generalized to other receptors and proteins in which metastable thermal equilibria have been identified and perturbed by ligand binding and downstream signaling.