The retinal structure of channelrhodopsin‐2 assessed by resonance Raman spectroscopy

Nack, Melanie; Radu, Ionela; Bamann, Christian; Bamberg, Ernst; Heberle, Joachim

doi:10.1016/j.febslet.2009.10.052

Cited by 64 publications

(171 citation statements)

References 35 publications

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“…The reason for this discrepancy is the invasive character of retinal extraction in the previously reported work (20). This process required breaking the Schiff base linker by subjecting the protein to EtOH followed by extraction of retinals into hexanes and HPLC purification (55).…”

Section: Resultsmentioning

confidence: 99%

“…Previously, a population of 30% 13-cis retinal based on retinal extraction with subsequent HPLC analysis and resonance Raman experiments has been reported (20).…”

Section: Resultsmentioning

confidence: 99%

“…In contrast to bacteriorhodopsin, no light adaption was observed using resonance Raman techniques (20) or visual spectroscopy (12). The occurrence of a conformer mixture in the ground state without light adaption would make ChR2 unique among the microbial retinal proteins, but additional data are needed to confirm these observations more directly at improved atomic resolution.…”

mentioning

confidence: 90%

“…The X-ray structure of the ChR chimera shows the retinal in an all-trans configuration (9), although other conformations cannot be excluded at the obtained resolution. Results of retinal extraction in conjunction with resonance Raman studies were interpreted as an isomer mixture containing 30% of a 13-cis retinal in dark-and light-adapted ChR2 (20). In addition, nanosecond IR spectroscopy on the E123T mutant of ChR2 indicated the presence of some 13-cis retinal in the dark state Significance Channelrhodopsin-2 is a dimeric membrane protein functioning as a light-gated ion channel, which has triggered numerous optogenetic applications.…”

mentioning

confidence: 99%

“…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.…”

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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.

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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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Section: Resultsmentioning

confidence: 99%

“…Previously, a population of 30% 13-cis retinal based on retinal extraction with subsequent HPLC analysis and resonance Raman experiments has been reported (20).…”

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 90%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

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.

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217

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Time‐Resolved and Surface‐Enhanced Infrared Spectroscopy

Heberle

2015

Pumps, Channels, and Transporters

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The Desensitized Channelrhodopsin‐2 Photointermediate Contains 13 ‐cis, 15 ‐syn Retinal Schiff Base

et al. 2021

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Channelrhodopsin-2 (ChR2) is al ight-gated cation channel and was used to laythe foundations of optogenetics.Its dark state X-raystructure has been determined in 2017 for the wild-type,w hichi st he prototype for all other ChR variants. However,t he mechanistic understanding of the channel function is still incomplete in terms of structural changes after photon absorption by the retinal chromophore and in the framework of functional models.H ence,d etailed information needs to be collected on the dark state as well as on the different photointermediates.F or ChR2 detailed knowledge on the chromophore configuration in the different states is still missing and ac onsensus has not been achieved. Using DNPenhanced solid-state MAS NMR spectroscopyo np roteoliposome samples,w eu nambiguously determined the chromophore configuration in the desensitized state,and we show that this state occurs towards the end of the photocycle.

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The retinal structure of channelrhodopsin‐2 assessed by resonance Raman spectroscopy

Cited by 64 publications

References 35 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

Time‐Resolved and Surface‐Enhanced Infrared Spectroscopy

The Desensitized Channelrhodopsin‐2 Photointermediate Contains 13 ‐cis, 15 ‐syn Retinal Schiff Base

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