An Active Photoreceptor Intermediate Revealed by In Situ Photoirradiated Solid-State NMR Spectroscopy

Tomonaga, Yuya; Hidaka, Tetsurou; Kawamura, Izuru; Nishio, Takudo; Ohsawa, Kazuhiro; Okitsu, Takashi; Wada, Akimori; Sudo, Yuki; Kamo, Naoki; Ramamoorthy, Ayyalusamy; Naito, Akira

doi:10.1016/j.bpj.2011.10.022

Cited by 29 publications

(48 citation statements)

References 19 publications

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“…Another option is to equip the DNP solid-state probe with a light guide and illuminate the sample directly while it is spinning in the stator. Such an approach has been shown by a number of laboratories using different designs with DNP (46) or standard MAS probes (62)(63)(64). The latter approach offers better temperature control, but quick freezing of the spinning samples is not possible.…”

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.

100

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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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.

100

217

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“…This large and specific shift is also observed in the protein moiety of bacteriorhodopsin [11][12][13] and sensory rhodopsin II. [10] It was also realized that the 13 C NMR intensity of a methyl carbon such as at position 20 is much higher than an olefinic carbon such as at position 15 (data not shown). Thus the retinal chromophore labeling with 13 C at position 20 provides a good reporter for investigating retinal configurations.…”

mentioning

confidence: 93%

“…[10] These intermediates are difficult to distinguish using visible spectroscopy. The 13 C NMR signals from [20-13 C]retinal-ppR and ppR/pHtrII revealed that multiple M-intermediates with 13-cis, 15-anti retinal configurations coexist under continuously photoirradiated conditions.…”

mentioning

confidence: 99%

“…The 13 C NMR signals from [20-13 C]retinal-ppR and ppR/pHtrII revealed that multiple M-intermediates with 13-cis, 15-anti retinal configurations coexist under continuously photoirradiated conditions. [10] It has been reported that the retinal chromophore 13 C-labeled at position 20 shows a significant chemical shift from about 14 to 22 ppm upon trans-cis photoisomerization. This is in contrast to other positions, such as positions 10, 11, and 19 [11] (Supporting Information, Table S1).…”

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confidence: 99%

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Color‐Discriminating Retinal Configurations of Sensory Rhodopsin I by Photo‐Irradiation Solid‐State NMR Spectroscopy

et al. 2014

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SRI (sensory rhodopsin I) can discriminate multiple colors for the attractant and repellent phototaxis. Studies aimed at revealing the color-dependent mechanism show that SRI is a challenging system not only in photobiology but also in photochemistry. During the photoreaction of SRI, an M-intermediate (attractant) transforms into a P-intermediate (repellent) by absorbing blue light. Consequently, SRI then cycles back to the G-state. The photoreactions were monitored with the (13)C NMR signals of [20-(13)C]retnal-SrSRI using in situ photo-irradiation solid-state NMR spectroscopy. The M-intermediate was trapped at -40 °C by illumination at 520 nm. It was transformed into the P-intermediate by subsequent illumination at 365 nm. These results reveal that the G-state could be directly transformed to the P-intermediate by illumination at 365 nm. Thus, the stationary trapped M- and P-intermediates are responsible for positive and negative phototaxis, respectively.

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“…6,7 Recently, we developed in-situ photo-irradiated solid-state NMR to induce the photo excitation and efficiently detected the photo-activated intermediate of a photoreceptor protein. 8 Bacteriorhodopsin (bR) in a purple membrane (PM) of H. salinarum reveals a light-driven proton pump during the photoisomerization of retinal. 9 The all-trans and 13-cis, 15-syn retinal configurations in the dark-adapted bR coexist in an isomeric ratio close to 1.…”

Section: Introductionmentioning

confidence: 99%

Change in local dynamics of bacteriorhodopsin with retinal isomerization under pressure as studied by fast magic angle spinning NMR

Kawamura

Yamaguchi

Nishikawa

et al. 2012

Polym J

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Bacteriorhodopsin (bR) has a retinal with all-trans and 13-cis, 15-syn configurations whose isomeric ratio is close to 1 in the dark-adapted bR, and the population of the 13-cis, 15-syn configuration can be increased under pressure. In our previous study, we applied pressure to bR using centrifugal force introduced by sample spinning and demonstrated that the 15 N nuclear magnetic resonance (NMR) signal intensity of the Schiff base in 13-cis, 15-syn retinal increased. In this study, we demonstrate a pressure-induced change in the local dynamics of the protein side in the vicinity of retinal. In the 13 C dipolar decoupling and magic angle-spinning NMR spectra of [3-13 C]Ala-labeled bR under fast spinning at 10 and 12 kHz, corresponding to 44 and 63 bar, respectively, the 13 C NMR signal at 16.6 p.p.m. appeared prominently as a sharp peak. This peak is assigned to Ala81 and Ala84 residues located in the vicinity of the retinal. It was observed that the change in the local dynamics in the vicinity of the retinal was caused by the applied pressure. These experiments demonstrate that fast magic angle spinning in NMR provides a pressure source for investigating the structure and change in the dynamics of biomacromolecules.

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An Active Photoreceptor Intermediate Revealed by In Situ Photoirradiated Solid-State NMR Spectroscopy

Cited by 29 publications

References 19 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

Color‐Discriminating Retinal Configurations of Sensory Rhodopsin I by Photo‐Irradiation Solid‐State NMR Spectroscopy

Change in local dynamics of bacteriorhodopsin with retinal isomerization under pressure as studied by fast magic angle spinning NMR

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