The Branched Photocycle of the Slow-Cycling Channelrhodopsin-2 Mutant C128T

Stehfest, Katja; Ritter, Eglof; Berndt, André; Bartl, Franz; Hegemann, Peter

doi:10.1016/j.jmb.2010.03.031

Cited by 66 publications

(82 citation statements)

References 41 publications

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“…Fig. 2B shows the 13 C-DNP-enhanced MAS NMR spectrum of ChR2. Using AMUPOL as the polarizing agent, a 62-fold signal enhancement was achieved under our experimental conditions.…”

Section: Resultsmentioning

confidence: 99%

“…Using AMUPOL as the polarizing agent, a 62-fold signal enhancement was achieved under our experimental conditions. The observed resonances mainly stem from the 13 C natural abundance background of protein, glycerol, and lipid. To suppress these signals and identify the 13 C-labeled retinal sites, a double quantum filter has been applied, revealing just two peaks from retinal carbons C14 and C15 at 126.3 and 166.5 ppm, respectively (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Enlightening the photoactive site of channelrhodopsin-2 by DNP-enhanced solid-state NMR spectroscopy

Becker‐Baldus

Bamann

Saxena

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

217

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Channelrhodopsin-2 from Chlamydomonas reinhardtii is a lightgated ion channel. Over recent years, this ion channel has attracted considerable interest because of its unparalleled role in optogenetic applications. However, despite considerable efforts, an understanding of how molecular events during the photocycle, including the retinal trans-cis isomerization and the deprotonation/reprotonation of the Schiff base, are coupled to the channel-opening mechanism remains elusive. To elucidate this question, changes of conformation and configuration of several photocycle and conducting/nonconducting states need to be determined at atomic resolution. Here, we show that such data can be obtained by solid-state NMR enhanced by dynamic nuclear polarization applied to 15 N-labeled channelrhodopsin-2 carrying 14,15-13 C 2 retinal reconstituted into lipid bilayers. In its dark state, a pure all-trans retinal conformation with a stretched C14-C15 bond and a significant out-of-plane twist of the H-C14-C15-H dihedral angle could be observed. Using a combination of illumination, freezing, and thermal relaxation procedures, a number of intermediate states was generated and analyzed by DNP-enhanced solid-state NMR. Three distinct intermediates could be analyzed with high structural resolution: the early P 500 1 K-like state, the slowly decaying late intermediate P 480 4 , and a third intermediate populated only under continuous illumination conditions. Our data provide novel insight into the photoactive site of channelrhodopsin-2 during the photocycle. They further show that DNP-enhanced solid-state NMR fills the gap for challenging membrane proteins between functional studies and X-ray-based structure analysis, which is required for resolving molecular mechanisms.ince their discovery (1), channelrhodopsins (ChRs) have generated enormous interest because of the rapid development of their applications in optogenetics (2-7). Commonly, ChR2 from Chlamydomonas reinhardtii (8) and its variants are used thanks to their favorable expression levels. They are the only proteins known today functioning as light-gated ion channels (Fig. 1A). Like other microbial retinal proteins, they undergo a periodic photocycle. In ChRs, this photocycle is coupled to channel opening and closing as revealed in electrophysiological recordings (8). A chimera of ChR1 and ChR2 has been crystallized to yield a structure at 2.3-Å resolution (9). However, little is known on how this coupling functions on a molecular level, and a large number of studies based on visible (10-13), IR (11,[14][15][16][17][18][19], resonance Raman spectroscopy (20, 21), and EPR spectroscopy (22, 23) has been performed to address this question.The photocycles of microbial rhodopsins are usually compared with bacteriorhodopsin, the first discovered and most studied lightdriven proton pump (24). Without any illumination, microbial retinal proteins thermally equilibrate into a dark state (25). In the case of bacteriorhodopsin, for example, this state contains a mixture of two species terme...

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“…Fig. 2B shows the 13 C-DNP-enhanced MAS NMR spectrum of ChR2. Using AMUPOL as the polarizing agent, a 62-fold signal enhancement was achieved under our experimental conditions.…”

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Enlightening the photoactive site of channelrhodopsin-2 by DNP-enhanced solid-state NMR spectroscopy

Becker‐Baldus

Bamann

Saxena

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

217

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“…11 and here renamed as ChR2 CA/C128T -C79/208 (formerly, ChR2-C79/208). The C128T substitution is known to prolong the conducting state and thus to enable the accumulation of P 3 520 under continuous illumination (9,10).…”

Section: Methodsmentioning

confidence: 99%

“…C128T, show a prolonged lifetime of the conducting state (8). These mutants are thus very well suited for trapping the P 3 520 state, the conducting open channel state (also named P520), in spectroscopic measurements, because no other effect except a prolonged open state was found upon mutation of Cys-128 (9,10).…”

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confidence: 97%

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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“…For instance, introduction of H134R mutation in the ChR2 sequence has yielded a variant with increased photocurrent amplitude (14), and further mutation of the Glu-123 residue to threonine led to faster off-kinetics (15). Other investigators have identified and modified those residues involved in ChR2 photocycle (15)(16)(17). Recently, the E123T/T159C double mutant that combines both large photocurrents and accelerated photocycle was generated (18).…”

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confidence: 99%

Bioinformatic and Mutational Analysis of Channelrhodopsin-2 Protein Cation-conducting Pathway

Plazzo

Franceschi

Broi

et al. 2012

Journal of Biological Chemistry

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Background: ChR2 is a light-gated ion channel allowing fast non-invasive control of cell membrane potential. Results: We combined bioinformatic modeling and electrophysiology to infer structure/function details on ChR2. Conclusion: We show a complete structural model of the channel, describe the ion-conducting pathway and identify key residues involved in ionic permeability and in photoactivation. Significance: These results expand our knowledge on the structural determinants of ChR2.

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The Branched Photocycle of the Slow-Cycling Channelrhodopsin-2 Mutant C128T

Cited by 66 publications

References 41 publications

Enlightening the photoactive site of channelrhodopsin-2 by DNP-enhanced solid-state NMR spectroscopy

Enlightening the photoactive site of channelrhodopsin-2 by DNP-enhanced solid-state NMR spectroscopy

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

Bioinformatic and Mutational Analysis of Channelrhodopsin-2 Protein Cation-conducting Pathway

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