Kinetic and Vibrational Isotope Effects of Proton Transfer Reactions in Channelrhodopsin-2

Abstract

Channelrhodopsins (ChRs) are light-gated cation channels. After blue-light excitation, the protein undergoes a photocycle with different intermediates. Here, we have recorded transient absorbance changes of ChR2 from Chlamydomonas reinhardtii in the visible and infrared regions with nanosecond time resolution, the latter being accomplished using tunable quantum cascade lasers. Because proton transfer reactions play a key role in channel gating, we determined vibrational as well as kinetic isotope effects (VIEs and KIEs) of carboxylic groups of various key aspartic and glutamic acid residues by monitoring their C=O stretching vibrations in H2O and in D2O. D156 exhibits a substantial KIE (>2) in its deprotonation and reprotonation, which substantiates its role as the internal proton donor to the retinal Schiff base. The unusual VIE of D156, upshifted from 1736 cm(-1) to 1738 cm(-1) in D2O, was scrutinized by studying the D156E variant. The C=O stretch of E156 shifted down by 8 cm(-1) in D2O, providing evidence for the accessibility of the carboxylic group. The C=O stretching band of E90 exhibits a VIE of 9 cm(-1) and a KIE of ∼2 for the de- and the reprotonation reactions during the lifetime of the late desensitized state. The KIE of 1 determined in the time range from 20 ns to 5 ms is incompatible with early deprotonation of E90.

Photoexcitation of the P4480 State Induces a Secondary Photocycle That Potentially Desensitizes Channelrhodopsin-2

“…This recovery time constant agrees with the time constant for the decay of P 4 480 to ChR 470 reported in the literature, τ = 6 -20 s, 10,14,15 confirming that the second photocycle of CrChR2 observed here originates from photo-excitation of P 4 480 . Note also the similarity of the data in Fig.…”

“…With τ ≈ 1.6 ms and τ ≈ 4.6 ms we resolve the rise and decay of the P 3 520 intermediate. 14 As expected, the amplitude of these two components decreases as Δt decreases, (Fig. 2B, compare blue and red curves).…”

Microbial Rhodopsins: Diversity, Mechanisms, and Optogenetic Applications

“…The central gate Glu90 deprotonates during the photocycle and, according to one view, this event initiates formation of the conductive pore (99, 115). However, other authors have proposed that deprotonation of Glu90 occurs only during the formation of the nonconductive P480 intermediate (97, 100). …”

“…Time-resolved FTIR spectroscopy has identified Asp156 (corresponding to Asp115 in BR; Figure 4, left) as the proton donor in Cr ChR2 (97, 100). However, this conclusion has been challenged by the observation that the kinetics of Asp156 deprotonation does not match the Schiff base reprotonation (87).…”

Atomistic Insight into the Role of Threonine 127 in the Functional Mechanism of Channelrhodopsin-2

“…Time-resolved UV/Vis experiments were performed with a commercial flash photolysis unit (LKS80, Applied Photophysics, Leatherhead, Surrey, UK), as described previously [17]. Briefly, a 10 ns laser pulse (Nd:YAG, Quanta-Ray, Spectra-Physics) tuned to 450 nm by an optical parametric oscillator (OPTA) was used to induce the photocycle.…”