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  • Pre-gating conformational changes in the ChETA variant of channelrhodopsin-2 monitored by nanosecond IR spectroscopy.

Pre-gating conformational changes in the ChETA variant of channelrhodopsin-2 monitored by nanosecond IR spectroscopy.

Journal of the American Chemical Society (2015-01-15)
Víctor A Lórenz-Fonfría, Bernd-Joachim Schultz, Tom Resler, Ramona Schlesinger, Christian Bamann, Ernst Bamberg, Joachim Heberle
摘要

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.

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Sigma-Aldrich
甘油-1,1,2,3,3-d5, 98 atom % D
Sigma-Aldrich
甘油-1,1,2,3,3-d5, endotoxin tested, 98 atom % D