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

Abstract

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 contrast to BR in which a downshift of 7–9 cm–1 occurs reflecting neutralization of the Asp85 counterion, and (iii) the CaChR1 protonated Schiff base (SB) has stronger hydrogen bonding than BR. A model is proposed to explain these results whereby at pH 7 the predominant counterion to the SB is Asp299 (the homologue to Asp212 in BR) while Glu169 (the homologue to Asp85 in BR) exists in a neutral state. We observe an unusual constancy of the resonance Raman spectra over the broad range from pH 9 to 2 and discuss its implications. These results are in accord with recent visible absorption and 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) Role of a helix B lysine residue in the photoactive site in channelrhodopsins. Biophys. J. 106, 1607–1617].

Proton Transfers in a Channelrhodopsin-1 Studied by Fourier Transform Infrared (FTIR) Difference Spectroscopy and Site-directed Mutagenesis

“…Our findings indicate that unlike the M 412 intermediate of BR, where Asp-85 is neutral and Asp-212 ionized, both homolog residues exist in neutral states in P 2 380 . On the basis of this and earlier measurements (8,10,11,21), a proton relay mechanism is suggested whereby: (i) a proton is transferred from Glu-169 to Asp-299 during the primary phototransition (P 1 formation) (11) and (ii) The positioning of residues is guided by known high resolution x-ray structure (12). The green arrow indicates the role of Asp-85 as an SB proton acceptor during M 412 formation (13).…”

“…A useful property of CaChR1 is that unlike the more extensively studied CrChR2, which possesses a mixture of all-trans and 13-cis retinal chromophore isomers in both its darkadapted and its light-adapted state (9), recent resonance Raman spectroscopy (RRS) and FTIR studies show that CaChR1 under our conditions has an almost pure all-trans retinal composition with a structure very similar to the light-adapted light-driven proton pump bacteriorhodopsin (BR) (10,11). This property is particularly useful to simplify analysis of structural changes occurring in ChRs, which in the case of CaChR1 appears to involve all-trans to 13-cis isomerization during the primary phototransition (11).…”

“…Expression, Purification, and Reconstitution of CaChR1 and Mutants-The expression, purification and reconstitution of wild-type and mutants of CaChR1 was recently described (10,11,21). Briefly, the seven-transmembrane domain of CaChR1 was expressed from Pichia pastoris in the presence of all-trans retinal, purified using a nickel-nitrilotriacetic acid-agarose affinity chromatography (Qiagen, Hilden, Germany), and reconstituted in Escherichia coli polar lipids (ECPL) with a mass ratio of 1:10 (CaChR1:ECPL).…”

“…Ultimately, this mechanism is reset in the late BR photocycle by the transfer of a proton from the cytoplasmic side of the membrane during N formation (Asp-96 deprotonates donating a proton to the SB), and Asp-85 deprotonates during O decay (resetting the proton release complex) (19,20). Recent studies (8,10,21) show that a different charge arrangement exists in the ground state photoactive site of CaChR1 as compared with BR (Fig. 1B).…”

The early development and application of FTIR difference spectroscopy to membrane proteins: A personal perspective

“…These studies were all conducted in collaboration with John Spudich, a leader in the microbial rhodopsin field at the University of Texas Health Science Center at Houston. They included FTIR difference studies along with RRS on halorhodopsin (HR) [197], sensory rhodopsins (SRI, SRII) from archaebacterial [29,31,34,38,184], fungal neurospora rhodopsin (NO) [30], anabaena sensory rhodopsin from cyanobacteria (ASR) [32], green and blue proteorhodopsin proton pumps from marine bacteria (PRs) [4,5,24,28,33,129,130]; archaerhodopsins-3 (AR3), a BR-like protein [56,216] and most recently channelrhodopsins (ChRs) from algae [173,174,176,250]. These proteins span a range of functions which are representative of diverse biomembranes process and include active anion transport (HRs), signal transduction (SRI, SRII and ASR), and light-gated ion channels (ChRs).…”