Structural Differences between the Closed and Open States of Channelrhodopsin-2 as Observed by Epr Spectroscopy

Krause, Nils; Engelhard, Christopher; Heberle, Joachim; Schlesinger, Ramona; Bittl, Robert

doi:10.1016/j.bpj.2013.11.1500

Cited by 7 publications

(13 citation statements)

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“…EPR spectroscopic measurements indicated that the dimers are tightly connected. 76,77 An oligomeric structure is not unusual for microbial rhodopsins, e.g. BR is organized as a trimer.…”

Section: General Structurementioning

confidence: 99%

“…157 Furthermore, cryo-electron microscopic (cryo-EM) and electron paramagnetic resonance (EPR) spectroscopic measurements revealed movements of TM2, TM6 and TM7 during formation of the conducting state. 76,77,165 77,165 The MD simulation that led to the conclusions of the EHT-model was based on the C1C2 structure in which E3 forms a hydrogen bond to Asn258. 41 In contrast, the more recently released during the 15-anti cycle.…”

Section: Photocycle Modelsmentioning

confidence: 99%

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

et al. 2019

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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

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“…EPR spectroscopic measurements indicated that the dimers are tightly connected. 76,77 An oligomeric structure is not unusual for microbial rhodopsins, e.g. BR is organized as a trimer.…”

Section: General Structurementioning

confidence: 99%

Section: Photocycle Modelsmentioning

confidence: 99%

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

et al. 2019

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“…Reprotonation of the RSB from the proton donor (D156) occurs simultaneously with channel closure during the decay of the conductive P 3 520 intermediate [7]. Electron paramagnetic resonance experiments on Cr ChR2 have shown that helices B and F move outwards upon formation of the conductive P 3 520 state [8]. Such helical movements persist even until the late desensitized P 4 480 state is formed [9].…”

Section: Introductionmentioning

confidence: 99%

Resonance Raman and FTIR spectroscopic characterization of the closed and open states of channelrhodopsin‐1

et al. 2014

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a b s t r a c tChannelrhodopsin-1 from Chlamydomonas augustae (CaChR1) is a light-activated cation channel, which is a promising optogenetic tool. We show by resonance Raman spectroscopy and retinal extraction followed by high pressure liquid chromatography (HPLC) that the isomeric ratio of all-trans to 13-cis of solubilized channelrhodopsin-1 is with 70:30 identical to channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2).

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“…Modern spin labeling applications also use orthogonal labeling schemes for proteins that contain functionally important cysteine side chains. 13,14 A summary of the rhodopsin studies is described below with discussions about the different directions of membrane protein EPR in the field. 8 and 9).…”

Section: Lydia N Caromentioning

confidence: 99%

Characterizing rhodopsin signaling by EPR spectroscopy: from structure to dynamics

Eps

Lydia

Morizumi

et al. 2015

Photochem Photobiol Sci

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Electron paramagnetic resonance (EPR) spectroscopy, together with spin labeling techniques, has played a major role in the characterization of rhodopsin, the photoreceptor protein and G protein-coupled receptor (GPCR) in rod cells. Two decades ago, these biophysical tools were the first to identify transmembrane helical movements in rhodopsin upon photo-activation, a critical step in the study of GPCR signaling. EPR methods were employed to identify functional loop dynamics within rhodopsin, to measure light-induced millisecond timescale changes in rhodopsin conformation, to characterize the effects of partial agonists on the apoprotein opsin, and to study lipid interactions with rhodopsin. With the emergence of advanced pulsed EPR techniques, the stage was set to determine the amplitude of structural changes in rhodopsin and the dynamics in the rhodopsin signaling complexes. Work in this area has yielded invaluable information about mechanistic properties of GPCRs. Using EPR techniques, receptors are studied in native-like membrane environments and the effects of lipids on conformational equilibria can be explored. This perspective addresses the impact of EPR methods on rhodopsin and GPCR structural biology, highlighting historical discoveries made with spin labeling techniques, and outlining exciting new directions in the field.

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Structural Differences between the Closed and Open States of Channelrhodopsin-2 as Observed by Epr Spectroscopy

Cited by 7 publications

References 0 publications

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

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

Resonance Raman and FTIR spectroscopic characterization of the closed and open states of channelrhodopsin‐1

Characterizing rhodopsin signaling by EPR spectroscopy: from structure to dynamics

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