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.febslet.2013.08.043

Cited by 60 publications

(87 citation statements)

References 32 publications

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“…By a combination of different time-resolved fluorescence techniques, we have presented results that lend support to the helix tilt model (5,(11)(12)(13) of helix B upon formation of the open channel state and gave insight in the possible mechanism by comparing the CrChR2 data with time-resolved anisotropy data from other retinal proteins. Accessibility studies revealed a pH-dependent structural heterogeneity close to the inner gate.…”

Section: Iafmentioning

confidence: 64%

“…A key role for helix B in light-induced channel opening and closing has been suggested. Evidence for light-induced movements of helix B is based on structural studies using electron crystallography and EPR spectroscopy (double electron-electron resonance) (11)(12)(13). Although double electron-electron resonance measurements suggest a light-induced displacement/outward tilt of helix B (11,13), electron crystallographic studies found additional evidence for a loss of order in that helix (12).…”

Section: Discussionmentioning

confidence: 99%

“…To detect dynamic structures and conformational changes of helix B via time-resolved fluorescence depolarization (16, 21), we selectively labeled position Cys-79 in a CrChR2 variant, in which the native cysteines, except for Cys-79 and Cys-208, were exchanged to alanine (11). Because the two remaining cysteines exhibit differential reactivity toward IAF, we were able to exclusively label position 79 at helix B (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Evidence for light-induced movements of helix B comes from structural studies using electron crystallography and EPR spectroscopy (double electronelectron resonance) (11)(12)(13). Together these data suggest that in contrast to bR and sensory rhodopsin, where large helix movements were observed for helix F upon reprotonation of the retinal Schiff base (14 -18), helix B in CrChR2 undergoes prominent light-induced rearrangements.…”

mentioning

confidence: 98%

“…However, the EPR distance measurements are not able to discriminate between conformational changes of the spin label and structural changes of the protein moiety. Therefore, it is not entirely clear whether the light-induced changes (11,13) originate from helix B displacements or local structural changes of the loop or label conformation.…”

mentioning

confidence: 99%

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Light and pH-induced Changes in Structure and Accessibility of Transmembrane Helix B and Its Immediate Environment in Channelrhodopsin-2

Volz

Krause

Balke

et al. 2016

Journal of Biological Chemistry

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A variant of the cation channel channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2) was selectively labeled at position Cys-79 at the end of the first cytoplasmic loop and the beginning of transmembrane helix B with the fluorescent dye fluorescein (acetamidofluorescein). We utilized (i) time-resolved fluorescence anisotropy experiments to monitor the structural dynamics at the cytoplasmic surface close to the inner gate in the dark and after illumination in the open channel state and (ii) time-resolved fluorescence quenching experiments to observe the solvent accessibility of helix B at pH 6.0 and 7.4. The light-induced increase in final anisotropy for acetamidofluorescein bound to the channel variant with a prolonged conducting state clearly shows that the formation of the open channel state is associated with a large conformational change at the cytoplasmic surface, consistent with an outward tilt of helix B. Furthermore, results from solute accessibility studies of the cytoplasmic end of helix B suggest a pH-dependent structural heterogeneity that appears below pH 7. At pH 7.4 conformational homogeneity was observed, whereas at pH 6.0 two protein fractions exist, including one in which residue 79 is buried. This inaccessible fraction amounts to 66% in nanodiscs and 82% in micelles. Knowledge about pH-dependent structural heterogeneity may be important for CrChR2 applications in optogenetics.Channelrhodopsins are involved in phototaxis and photophobia of unicellular green algae (1). They constitute a new class of light-gated ion channels containing the seven-transmembrane helix motif and the chromophore retinal as the light-sensitive cofactor, which are both common to the other retinal-containing proteins such as bacteriorhodopsin (bR) 4 or visual rhodopsin (2). In CrChR2 the chromophore retinal is bound to Lys-257 (3). Channelrhodopsin activation via lightinduced isomerization of retinal from all-trans to 13-cis is coupled to a transient cofactor deprotonation and a functional protein structural change. In channelrhodopsin, as well as in other retinal-containing photoreceptors, subtle changes in the chromophore vicinity trigger large scale protein conformational changes remote from the chromophore binding pocket. Rearrangement of helix B is hypothesized to constitute a key element in channel opening by allowing the entry of water molecules (4 -6). A continuous water wire is a prerequisite for the formation of the ion-conducting pathway. A hydrophilic pore between helices A, B, C, and G was suggested to serve as the ion permeation pathway based on the high resolution crystal structure of the C1C2 chimera (3 10).The inner gate was hypothesized to be involved in CrChR2 activation (formation of the open conducting channel state) together with the tilt of helix B (5). Evidence for light-induced movements of helix B comes from structural studies using electron crystallography and EPR spectroscopy (double electronelectron resonance) (11-13). Together these data suggest that in contrast to bR and sensory rhod...

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Section: Iafmentioning

confidence: 64%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 98%

mentioning

confidence: 99%

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Light and pH-induced Changes in Structure and Accessibility of Transmembrane Helix B and Its Immediate Environment in Channelrhodopsin-2

Volz

Krause

Balke

et al. 2016

Journal of Biological Chemistry

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Die frühe Entstehung der ionenleitenden Pore in Channelrhodopsin‐2

et al. 2014

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Kanalrhodopsine (Channelrhodopsins,C hRs) sind lichtgesteuerte Ionenkanäle,d ie zahlreiche Anwendungen in der Optogenetik finden. Sie ermçglichen die genaue Kontrolle neuronaler Aktivität durch Licht.H ier entwickeln wir auf der Basis zeitaufgelçster FTIR-Spektroskopie ein Modell für die frühe Bildung der ionenleitenden Pore auf molekularer Ebene. Die Photo-Isomerisierung des Retinal-Chromophors führt zu einer Abwärtsbewegung der hochkonservierten Aminosäure E90;dies çffnet die Pore.Moleküldynamik(MD-Simulationen zeigen das Eindringen von Wassermolekülen durch diese Öffnung in das hydrophobe Vestibül oberhalb der Pore.D adurch kippt Helix 2, und der Kanal çffnet sich vollständig innerhalb von Millisekunden. Da E90 in ChRs hochkonserviert ist, kçnnte das hier vorgestellte "E90-Helix2-tilt(EHT)-Modell" einen generellen Aktivierungsmechanismus darstellen. Es erçffnet einen neuen Ansatz für weitere mechanistische Studien sowied ie Mçglichkeit, den Kanal für spezifischeA nwendungen maßzuschneidern.

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Triggered Functional Dynamics of AsLOV2 by Time‐Resolved Electron Paramagnetic Resonance at High Magnetic Fields

et al. 2023

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We present time‐resolved Gd−Gd electron paramagnetic resonance (TiGGER) at 240 GHz for tracking inter‐residue distances during a protein's mechanical cycle in the solution state. TiGGER makes use of Gd‐sTPATCN spin labels, whose favorable qualities include a spin‐7/2 EPR‐active center, short linker, narrow intrinsic linewidth, and virtually no anisotropy at high fields (8.6 T) when compared to nitroxide spin labels. Using TiGGER, we determined that upon light activation, the C‐terminus and N‐terminus of AsLOV2 separate in less than 1 s and relax back to equilibrium with a time constant of approximately 60 s. TiGGER revealed that the light‐activated long‐range mechanical motion is slowed in the Q513A variant of AsLOV2 and is correlated to the similarly slowed relaxation of the optically excited chromophore as described in recent literature. TiGGER has the potential to valuably complement existing methods for the study of triggered functional dynamics in proteins.

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Structural differences between the closed and open states of channelrhodopsin‐2 as observed by EPR spectroscopy

Cited by 60 publications

References 32 publications

Light and pH-induced Changes in Structure and Accessibility of Transmembrane Helix B and Its Immediate Environment in Channelrhodopsin-2

Light and pH-induced Changes in Structure and Accessibility of Transmembrane Helix B and Its Immediate Environment in Channelrhodopsin-2

Die frühe Entstehung der ionenleitenden Pore in Channelrhodopsin‐2

Triggered Functional Dynamics of AsLOV2 by Time‐Resolved Electron Paramagnetic Resonance at High Magnetic Fields

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