Resonance Raman and Low Temperature FTIR Characterization of the Red Shifted Channelrhodopsin 1 from Chlamydomonas Augustae

Abstract

Channelrhodopsins (ChRs) control phototaxis in green algae and function as light-gated cation channels when expressed in animal cells. Because ChRs can be functionally expressed in neuronal membranes, this distinct family of microbial rhodopsins have rapidly become an important tool in neuroscience. While the light-activated molecular changes occurring in channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2) have been extensively studied, little is known about such changes in the diverse groups of other ChRs including the major class of channelrhodopsin-1 (ChR1). Here, we have characterized the structure and molecular changes in ChR1 from Chlamydomonas augustae (CaChR1). This ChR has properties advantageous for light modulated neuronal control including a red-shifted λmax and slow light inactivation compared to CrChR2 (Hou, S. et al. (2012) Photochem Photobiol 88, 119-128). Near-infrared confocal resonance Raman spectroscopy (RRS) reveals that in contrast to CrChR2, which contains a mixture of retinal isomers, the retinal chromophore structure of CaChR1 is almost completely all-trans in the light-adapted state similar to many microbial rhodopsins including bacteriorhodopsin. In addition, unlike other ChR1s, such as CrChR1 (ChR1 from Chlamydomonas reinhardtii), which exhibit significant shifts in λmax at different pHs, the RRS of CaChR1 including the ethylenic frequency which reflects λmax remains largely unaltered over a wide pH range. This insensitivity to pH is despite the presence of the residue Glu87 (CrChR1 sequence numbering) which has been previously associated with pH sensitivity. Low-temperature FITR difference-spectroscopy reveals that the primary phototransition of CaChR1 to the K photointermediate involves an all-trans to 13-cis retinal isomerization and significant changes in the protein structure including structural changes in the peptide backbone, Asp/Glu residues, Cys residues and internal water molecules. These results are discussed in terms of possible differences in the molecular mechanism of various ChRs.