Retinal Chromophore Structure and Schiff Base Interactions in Red-ShiftedChannelrhodopsin-1 from Chlamydomonas augustae

摘要

Channelrhodopsins (ChRs), which form a distinct branch of the microbial rhodopsin family, control phototaxis in green algae. Because ChRs can be expressed and function in neuronal membranes as light-gated cation channels, they have rapidly become an important optogenetic tool in neurobiology. While channelrhodopsin-2 from the unicellular alga Chlamydomonas reinhardtii (CrChR2) is the most commonly used and extensively studied optogenetic ChR, little is known about the properties of the diverse group of other ChRs. In this study, near-infrared confocal resonance Raman spectroscopy along with hydrogen–deuterium exchange and site-directed mutagenesis were used to study the structure of red-shifted ChR1 from Chlamydomonas augustae (CaChR1). These measurements reveal that (i) CaChR1 has an all-trans-retinal structure similar to those of the light-driven proton pump bacteriorhodopsin (BR) and sensory rhodopsin II but different from that of the mixed retinal composition of CrChR2, (ii) lowering the pH from 7 to 2 or substituting neutral residues for Glu169 or Asp299 does not significantly shift the ethylenic stretch frequency more than 1–2 cm^–1 in contrastto BR in which a downshift of 7–9 cm^–1 occursreflecting neutralization of the Asp85 counterion, and (iii) the CaChR1 protonated Schiff base (SB) has stronger hydrogenbonding than BR. A model is proposed to explain these results wherebyat pH 7 the predominant counterion to the SB is Asp299 (the homologueto Asp212 in BR) while Glu169 (the homologue to Asp85 in BR) existsin a neutral state. We observe an unusual constancy of the resonanceRaman spectra over the broad range from pH 9 to 2 and discuss itsimplications. These results are in accord with recent visible absorptionand current measurements of CaChR1 [Sineshchekov,O. A., et al. (2013) Intramolecular proton transfer in channelrhodopsins. Biophys. J. 104, 807–817; Li, H., et al. (2014) Roleof a helix B lysine residue in the photoactive site in channelrhodopsins. Biophys. J. 106, 1607–1617].