Retinal isomer ratio in dark-adapted purple membrane and bacteriorhodopsin monomers

Scherrer, P; Mathew, M. K.; Sperling, W.; Stoeckenius, Walther

doi:10.1021/bi00428a063

Cited by 146 publications

(159 citation statements)

References 43 publications

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“…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). For bacteriorhodopsin, the results obtained in this way (56) agree well with data from other methods (26,27).…”

Section: Resultssupporting

confidence: 68%

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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Section: Resultssupporting

confidence: 68%

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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“…(Scherrer et al, 1989). This observation is consistent with the report by AIshuth et al (1984) that the population of 13-cis bR is greatly increased in dehydrated purple membrane samples.…”

Section: Materials and Methods: Gravimetricsupporting

confidence: 93%

Trapping the M sub 1 and M sub 2 substrates of bacteriorhodopsin for electron diffraction studies

Perkins¹

1992

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“…S8). In BR, dark adaptation corresponds to conversion from all-trans retinal to a mixture of all-trans and 13-cis retinal (34).…”

Section: Resultsmentioning

confidence: 99%

Mechanism of voltage-sensitive fluorescence in a microbial rhodopsin

Maclaurin¹,

Venkatachalam

Lee

et al. 2013

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

139

186

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Here, we present a detailed spectroscopic characterization of Archaerhodopsin 3 (Arch). We performed fluorescence spectroscopy on Arch and its photogenerated intermediates in Escherichia coli and in single HEK293 cells under voltage-clamp conditions. These experiments probed the effects of time-dependent illumination and membrane voltage on absorption, fluorescence, membrane current, and membrane capacitance. The fluorescence of Arch arises through a sequential three-photon process. Membrane voltage modulates protonation of the Schiff base in a 13-cis photocycle intermediate (M ⇌ N equilibrium), not in the ground state as previously hypothesized. We present experimental protocols for optimized voltage imaging with Arch, and we discuss strategies for engineering improved rhodopsin-based voltage indicators.

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Retinal isomer ratio in dark-adapted purple membrane and bacteriorhodopsin monomers

Cited by 146 publications

References 43 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

Trapping the M sub 1 and M sub 2 substrates of bacteriorhodopsin for electron diffraction studies

Mechanism of voltage-sensitive fluorescence in a microbial rhodopsin

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