Near-Ir Resonance Raman and Uv-Visible Absorption Kinetic Spectroscopy of Optogenetic Archaerhodopsin Neuronal Silencers: Effects of Membrane Potential

Abstract

Archaerhodopsin 3 (AR3) and Archaerhodopsin T (ArchT) are light-driven proton pumps that are used as genetically targetable neuronal silencers and fluorescent sensors of transmembrane potential. Unlike the more extensively studied bacteriorhodopsin (BR) from Halobacterium salinarum, AR3 and ArchT incorporate into the plasma membranes of E. coli and mammalian cells. We used near-IR resonance Raman confocal microscopy and time resolved UV-Vis pump-probe absorption spectroscopy to study the effects of pH and membrane potential on the retinal chromophore structure and photocycle kinetics of these proteins. Measurements were performed on AR3 and ArchT reconstituted into E. coli polar lipids and in vivo in E. coli expressing AR3. The retinal chromophore structure of AR3 is an all-trans configuration almost identical to BR over pH ranging from 3-11. Small changes detected in the retinal ethylenic stretching frequency and Schiff base (SB) hydrogen-bonding strength relative to BR may be related to an alternate water structure near the SB. In the case of the mutant D95N, an all-trans retinal O-like species (Oall-trans) is found at neutral pH. At higher pH, a 13-cis retinal N-like species (N13-cis) is detected which is attributed to a slowly decaying intermediate in the D95N red-light photocycle. However, the amount of N13-cis detected is reduced in E. coli but restored upon dissipation of the normal negative membrane potential. We postulate that these changes are due to the effect of membrane potential on the N13-cis to M13-cis equilibrium in the D95N red-light photocycle and on a molecular level by the effects of the electric field on the protonation/deprotonation of the cytoplasmic accessible SB. This mechanism offers a possible explanation for the observed dependence on transmembrane potential of the fluorescence of AR3, ArchT, and other microbial rhodopsins.