Vibrational Studies of Channelrhodopsin-1 from Chlamydomonas Augustae: Protonation Changes during the Early Photocycle

Abstract

Channelrhodopsins (ChRs) from green algae function as light-gated ion channels when expressed heterologously in mammalian cells. Considerable interest has focused on understanding the molecular mechanisms of ChRs in order to bioengineer their properties for specific optogenetic applications. Most studies have focused so far on channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2). Here, low-temperature and time-resolved rapid scan FTIR-difference spectroscopy, along with kinetic UV-Visible transient absorbance spectroscopy, are applied to study protein conformational changes occurring during the photocycle of the red-shifted channelrhodopsin-1 from Chlamydomonas augustae (CaChR1). Substitutions with isotope-labeled retinals or the retinal analog A2, site-directed mutagenesis, hydrogen-deuterium exchange and H218O substitution are used to assign bands to the retinal chromophore, protein, and internal water molecules. The primary phototransition of CaChR1 at 80 K involves, in contrast to CrChR2, almost exclusively all-trans to 13-cis isomerization of the retinal chromophore, similar to the primary phototransition of bacteriorhodopsin (BR). A negative amide II band is identified in the retinal ethylenic stretch region of CaChR1 which, along with amide I bands, reflects alterations in protein backbone structure early in the photocycle. A decrease in the hydrogen bond strength of a weakly hydrogen bonded internal water is detected in both CaChR1 and CrChR2, but the bands are much broader in CrChR2 indicating a more heterogeneous environment. Based on the data from mutations involving the residues Glu169 and Asp299 (homologs of the Asp85 and Asp212 Schiff base counterions in BR), as well as Phe139 (Lys132 in the C1C2 chimera located within hydrogen bonding distance of the homologs of Asp85 and Asp212), we propose a model for protonation changes occurring near the Schiff base during the P1 and P2 steps of the photocycle.