Conformational Changes of Channelrhodopsin-2

Abstract

Channelrhodopsin-2 (ChR2) is a member of the new class of light-gated ion channels which serve as phototaxis receptors in the green alga Chlamydomonas reinhardtii. The protein is employed in optogenetics where neural circuits are optically stimulated under high spatiotemporal control. Despite its rapidly growing use in physiological experiments, the reaction mechanism of ChR2 is poorly understood. Here, we applied vibrational spectroscopy to trace structural changes of ChR2 after light-excitation of the retinal chromophore. FT-IR difference spectra of the various photocycle intermediates revealed that stages of the photoreaction preceding (P(1) state) and succeeding (P(4)) the conductive state of the channel (P(3)) are associated with large conformational changes of the protein backbone as indicate by strong differences in the amide I bands. Critical hydrogen-bonding changes of protonated carboxylic amino acid side chains (D156, E90) were detected and discussed with regard to the functional mechanism. We used the C128T mutant where the lifetime of P(3) is prolonged and applied FT-IR and resonance Raman spectroscopy to study the conductive P(3) state of ChR2. Finally, a mechanistic model is proposed that links the observed structural changes of ChR2 to the changes in the channel's conductance.

Changes in the hydrogen-bonding strength of internal water molecules and cysteine residues in the conductive state of channelrhodopsin-1

“…The three negative bands in Fig. 1 at 1237 cm −1 , 1202 cm −1 , and 1163 cm −1 are assigned to C-C stretching vibrations of protonated all-trans retinal, 55 indicating that the photocycle in CaChR1 starts predominantly from the isomerization of the all-trans retinal, as in CrChR2 56,57 and in most microbial rhodopsins. 39 The IR intensity of the C-C stretching vibrations of the retinal is negligible unless the SB is protonated.…”

The light‐driven sodium ion pump: A new player in rhodopsin research

“…Although a number of recent studies have given us a hint about the dynamic properties of KR2 [24,33,54], we know almost nothing about (1) the detailed structural changes in the Schiff base region À including the formation of transient Na þ binding site in the O-intermediate À and (2) the large conformational change of the protein backbone during the photocycle. Similar to the cases of BR and HR [55][56][57][58], structural and spectroscopic techniques such as time-resolved (TR) macromolecular crystallography (e.g. Laue method and TR-serial femtosecond crystallography) and FTIR spectroscopy are suitable to study detailed structural changes inside the protein.…”

Enlightening the photoactive site of channelrhodopsin-2 by DNP-enhanced solid-state NMR spectroscopy

“…A chimera of ChR1 and ChR2 has been crystallized to yield a structure at 2.3-Å resolution (9). However, little is known on how this coupling functions on a molecular level, and a large number of studies based on visible (10-13), IR (11,[14][15][16][17][18][19], resonance Raman spectroscopy (20, 21), and EPR spectroscopy (22, 23) has been performed to address this question.The photocycles of microbial rhodopsins are usually compared with bacteriorhodopsin, the first discovered and most studied lightdriven proton pump (24). Without any illumination, microbial retinal proteins thermally equilibrate into a dark state (25).…”

“…A chimera of ChR1 and ChR2 has been crystallized to yield a structure at 2.3-Å resolution (9). However, little is known on how this coupling functions on a molecular level, and a large number of studies based on visible (10-13), IR (11,(14)(15)(16)(17)(18)(19), resonance Raman spectroscopy (20,21), and EPR spectroscopy (22,23) has been performed to address this question.…”