title: Pre-gating conformational changes in the ChETA variant of channelrhodopsin-2 monitored by nanosecond IR spectroscopy.
abstract: Light-gated ion permeation by channelrhodopsin-2 (ChR2) relies on the photoisomerization of the retinal chromophore and the subsequent photocycle, leading to the formation (on-gating) and decay (off-gating) of the conductive state. Here, we have analyzed the photocycle of a fast-cycling ChR2 variant (E123T mutation, also known as ChETA), by time-resolved UV/vis, step-scan FT-IR, and tunable quantum cascade laser IR spectroscopies with nanosecond resolution. Pre-gating conformational changes rise with a half-life of 200 ns, silent to UV/vis but detected by IR spectroscopy. They involve changes in the peptide backbone and in the H-bond of the side chain of the critical residue D156. Thus, the P1(500) intermediate must be separated into early and late states. Light-adapted ChR2 contains a mixture of all-trans and 13-cis retinal in a 70:30 ratio which are both photoactive. Analysis of ethylenic and fingerprint vibrations of retinal provides evidence that the 13-cis photocycle recovers in 1 ms. This recovery is faster than channel off-gating and most of the proton transfer reactions, implying that the 13-cis photocycle is of minor functional relevance for ChR2.
authors.affiliation: Experimental Molecular Biophysics, Department of Physics, Freie Universität Berlin , Arnimallee 14, 14195 Berlin, Germany.
authors.forename: Víctor A
authors.initials: VA
authors.lastname: Lórenz-Fonfría
authors.forename: Bernd-Joachim
authors.initials: BJ
authors.lastname: Schultz
authors.forename: Tom
authors.initials: T
authors.lastname: Resler
authors.forename: Ramona
authors.initials: R
authors.lastname: Schlesinger
authors.forename: Christian
authors.initials: C
authors.lastname: Bamann
authors.forename: Ernst
authors.initials: E
authors.lastname: Bamberg
authors.forename: Joachim
authors.initials: J
authors.lastname: Heberle
mesh_terms: D003624:Darkness; D004224:Diterpenes; D007700:Kinetics; D009154:Mutation; D010782:Photolysis; D011487:Protein Conformation; D012172:Retinaldehyde / Q000737:chemistry / Q000378:metabolism; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017550:Spectroscopy, Fourier Transform Infrared; D055672:Static Electricity; D013997:Time Factors; D014732:Vibration
chemical_list: D004224:Diterpenes; C031338:13-cis-retinal; D012243:Rhodopsin; D012172:Retinaldehyde