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

Abstract

Channelrhodopsins (ChRs) belong to the unique class of light-gated ion channels. The structure of channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2) has been resolved, but the mechanistic link between light-induced isomerization of the chromophore retinal and channel gating remains elusive. Replacements of residues C128 and D156 (DC gate) resulted in drastic effects in channel closure. T127 is localized close to the retinal Schiff base and links the DC gate to the Schiff base. The homologous residue in bacteriorhodopsin (T89) has been shown to be crucial for the visible absorption maximum and dark–light adaptation, suggesting an interaction with the retinylidene chromophore, but the replacement had little effect on photocycle kinetics and proton pumping activity. Here, we show that the T127A and T127S variants of CrChR2 leave the visible absorption maximum unaffected. We inferred from hybrid quantum mechanics/molecular mechanics (QM/MM) calculations and resonance Raman spectroscopy that the hydroxylic side chain of T127 is hydrogen-bonded to E123 and the latter is hydrogen-bonded to the retinal Schiff base. The C=N–H vibration of the Schiff base in the T127A variant was 1674 cm−1, the highest among all rhodopsins reported to date. We also found heterogeneity in the Schiff base ground state vibrational properties due to different rotamer conformations of E123. The photoreaction of T127A is characterized by a long-lived P2380 state during which the Schiff base is deprotonated. The conservative replacement of T127S hardly affected the photocycle kinetics. Thus, we inferred that the hydroxyl group at position 127 is part of the proton transfer pathway from D156 to the Schiff base during rise of the P3530 intermediate. This finding provides molecular reasons for the evolutionary conservation of the chemically homologous residues threonine, serine, and cysteine at this position in all channelrhodopsins known so far.

NeoR, a near-infrared absorbing rhodopsin

“…The D140 homolog position occupied by Thr in many microbial rhodopsins has been widely disregarded by experimentalists but was suggested by theoreticians to contribute to the proton transfer from RSBH + to the acceptor Asp or Glu in rhodopsin pumps and channels [33][34][35] . Moreover, replacement of this Thr in the proton pump Arch to Cys, T99C, in combination with the D95E mutation of the primary proton acceptor shifted the absorption from 556 nm to 626 nm thus creating the largest bathochromic color shift and the most red-shifted microbial rhodopsin which, however, remained unexplained 21 .…”

Multiscale QM/MM modelling of catalytic systems with ChemShell

“…165 QM/MM methods are now widely applied to the study of excited electronic states, in tandem with electronic structure software packages capable of time-dependent density functional theory or wavefunction-based excited state calculations. Light-sensitive proteins that have been simulated with Chem-Shell in recent studies include a wide range of rhodopsins, [166][167][168][169][170][171][172][173] photosystem II, [174][175][176] calcium-regulated photoproteins, 177 green fluorescent protein, 178 the phototoxic protein KillerRed, 179 bacterial phytochromes, 180,181 and cyanobacteriochromes. [182][183][184] Excited state calculations are typically used to simulate experimental UV-vis spectra, 185 and so are useful for characterising any molecular system containing a chromophore, including catalytic intermediates.…”