Tracking Pore Hydration in Channelrhodopsin by Site-Directed Infrared-Active Azido Probes

Abstract

Channelrhodopsins (ChRs) are retinal binding membrane proteins found in single-cell algae. Photoisomerization of ChRs leads to formation of an ion channel. The resulting change in membrane voltage modulates flagellate motions allowing phototaxis and photophobic responses. Heterologously expressed in host cells, ChRs allow the evocation or suppression of changes in membrane potential with high spatio-temporal resolution -this method has become known as optogenetics. Functional studies have raised questions concerning the molecular determinants for absorption, formation and closing of the ion channel and ion selectivity. It was the scope of this thesis to address these questions based on a comparison of the three different ChR variants C1C2, ReaChR (Red-activatable ChR) and Chrimson. C1C2 (λmax ≈ 470 nm) is a chimera of the Chlamydomonas reinhardtii ChRs CrChR1 and CrChR2. ReaChR (λmax ≈ 520 nm) is a variant of Volvox carteri ChR1 whose red-shifted absorption allows its use in deeper layers of organic tissue in optogenetic experiments. The even further red-shifted (Cs)Chrimson (λmax ≈ 590 nm) is a more distantly related ChR from Chlamydomonas noctigama with the N-terminal sequence from Chloromonas subdivisa ChR that is significantly more proton-selective. The photoreaction mechanism was investigated using FTIR (Fourier Transform Infrared) spectroscopy at room temperature and at cryostatic conditions. The results were complemented by UV-Vis spectroscopy and retinal extraction and subsequent HPLC (High Performance Liquid Chromatography) analysis.As in most microbial rhodopsins, the retinal cofactor in ChRs is predominantly in 13-trans,15anti conformation and bound to the protein by a retinal Schiff base (RSB) linkage to a lysine.Usually, the RSB is protonated in the dark (RSBH + ) stabilized by the counter-ion complex formed by a glutamate (counter-ion 1, Ci1) and an aspartate (counter-ion 2, Ci2).Photoreceptors are optimized to use photon energy to drive conformational changes of the protein backbone. Therefore, a fraction of the photon energy is stored by a transient distortion of the chromophore and separation of the charges in the active site by increased distance between the RSBH + and its counter-ions. In this thesis, it is shown that in ReaChR the transfer of the stored energy to the protein is largely affected by the Ci1 (Glu163) protonation state, being decelerated by protonated Ci1 due to an enhanced rigidity of the active site that stabilizes the distorted chromophore conformation. Instead, in Chrimson the chromophore

Genetically encoded non-canonical amino acids reveal asynchronous dark reversion of chromophore, backbone and side-chains in EL222

“…Our results indicate that CNF residues inserted in selected positions of EL222 can, after a careful scrutiny, provide residue-specific information on the photocycle not attainable by other probes naturally present in EL222, like the FMN chromophore or the protein backbone. This observation is in line with previous studies on other photoreceptors that, in contrast to our work, focused on a relatively low number of unnatural residues (typically less than ten) or did not comprehensively address their potential perturbation (23,24,(28)(29)(30)58). Based on our results with a large collection of mutants (45 for a protein of 210 residues), a major caveat of genetically encoded non-canonical side-chains as site-specific infrared reporters is the risk of mis-or over-interpretations when only a limited number of such probes are tested.…”

Local Electric Field Changes during the Photoconversion of the Bathy Phytochrome Agp2

“…In this context it is interesting to refer to the recent study by Kurttila et al who introduced para -azidophenylalanine at different positions in the prototypical phytochrome Dr BphP from Deinococcus radiodurans, among them were also the analogous positions studied in this work, i.e., Phe203 (Phe192 in Agp2) and Tyr176 (Tyr165 in Agp2) . The frequencies of the azide stretching modes respond to changes of the molecular environment, however, much less sensitive to variations of the electric field in a quantifiable fashion. − Instead, the azide modes are considered as rather qualitative sensors for polarity changes. Nevertheless, for Dr BphP, a significant downshift of the azide stretching mode from Pr → Pfr was reported and interpreted in terms of an increased polarity in Pfr .…”

Infrared Difference Spectroscopy of Proteins: From Bands to Bonds

“…By using the spectrally isolated band from (N 3 )­Phe at ∼2100 cm –1 and its sensitivity toward polarity changes, the authors deduced hydration changes at various sites of the putative ion permeation pore. Thanks to these probes they could spatially localized a hydration event in ReaChR that correlates with ion conductivity . In a final example, (N 3 )­Phe was incorporated in place of a Met and a Trp near the FAD of the BLUF photoreceptor PixD and AppA .…”

“…Combined with results obtained with other labeled sites, they concluded that helix F rotates in Meta I, which brings the azido group from a hydrophilic to a hydrophobic environment, a conformational change preceding the formation of the active state of the receptor in Meta II. More recently, Krause et al 761 also introduced site-specifically the unnatural amino acid (N 3 )Phe at different positions of the channelrhodopsin ReaChR. They used the introduced azide probe as a local polarity sensor, aiming to probe the formation and collapse of the ion-conducting pore.…”

“…Thanks to these probes they could spatially localized a hydration event in ReaChR that correlates with ion conductivity. 761 In a final example, (N 3 )Phe was incorporated in place of a Met and a Trp near the FAD of the BLUF photoreceptor PixD and AppA. 762 Vibrational changes from the azido group were measured both under static and under time-resolved conditions, the latter with ps time resolution.…”