Pre-Gating Conformational Changes in the ChETA Variant of Channelrhodopsin-2 Monitored by Nanosecond IR Spectroscopy

Lórenz-Fonfrı́a, Vı́ctor A.; Schultz, Bernd-Joachim; Resler, Tom; Schlesinger, Ramona; Bamann, Christian; Bamberg, Ernst; Heberle, Joachim

doi:10.1021/ja5108595

Cited by 39 publications

(80 citation statements)

References 82 publications

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“…using a similar spectroscopic assignment as in the resonance Raman study (39). In contrast to bacteriorhodopsin, no light adaption was observed using resonance Raman techniques (20) or visual spectroscopy (12).…”

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confidence: 69%

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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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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confidence: 69%

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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“…2C. Bands at 1,762(+), 1,745(+), and 1,737(−) cm −1 are from the terminal C=O of D156 (19,38,39), and bands at 1,728(+) and 1,717(−) cm −1 from E90 (19,40). Noteworthy is that none of the above bands showed a bell-shaped evolution in logarithmic time scale akin to the photocurrents.…”

Section: Resultsmentioning

confidence: 95%

Temporal evolution of helix hydration in a light-gated ion channel correlates with ion conductance

Lórenz-Fonfrı́a

Bamann

Resler

et al. 2015

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

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106

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The discovery of channelrhodopsins introduced a new class of lightgated ion channels, which when genetically encoded in host cells resulted in the development of optogenetics. Channelrhodopsin-2 from Chlamydomonas reinhardtii, CrChR2, is the most widely used optogenetic tool in neuroscience. To explore the connection between the gating mechanism and the influx and efflux of water molecules in CrChR2, we have integrated light-induced time-resolved infrared spectroscopy and electrophysiology. Cross-correlation analysis revealed that ion conductance tallies with peptide backbone amide I vibrational changes at 1,665(−) and 1,648(+) cm. These two bands report on the hydration of transmembrane α-helices as concluded from vibrational coupling experiments. Lifetime distribution analysis shows that water influx proceeded in two temporally separated steps with time constants of 10 μs (30%) and 200 μs (70%), the latter phase concurrent with the start of ion conductance. Water efflux and the cessation of the ion conductance are synchronized as well, with a time constant of 10 ms. The temporal correlation between ion conductance and hydration of helices holds for fast (E123T) and slow (D156E) variants of CrChR2, strengthening its functional significance.channelrhodopsin | optogenetics | channel gating | infrared spectroscopy | time-resolved spectroscopy I on channels are membrane proteins that mediate the passive movement of cations and anions across biological membranes, a process not only central for most living organisms but key to electrical excitability of cells. Upon activation, ion channels constitute a transient pathway to facilitate the permeation of ions across the cellular membrane. Ion channels can be switched, or gated, from a closed (nonconductive) to an open (conductive) state by external stimuli, such as ligands, voltage, or mechanical stress (1-3). Available structural information indicates that the ion-conducting pathway generally comprises a pore with wide regions solvated by water and constrictions sites formed by specific polar groups that confer ion selectivity (4). In the nonconductive state, the permeation of ions is prevented by energy barriers along the pore, either from physical occlusion or from the hydrophobic nature of residues lining the pore: the pore does not need to be physically occluded to be functionally closed (5). Habitual suspects for the gating mechanism, i.e., how the protein transits from the nonconductive to the conductive state, are diverse types of structural changes, such as orientation/rotation changes of transmembrane helices or of bulky (aromatic) side chains (6). Several computational studies have suggested a more subtle gating mechanism, where the thermodynamics and kinetics of the hydration of the ion-conducting pore might play a vital role (5,7,8). However, experimental support for this last proposal relies largely on studies on synthetic nanometric pores (9).Channelrhodopsins (ChRs) belong to the new class of lightgated ion channels, i.e., ion channels activated by light...

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“…Both channels were digitized by picoscopes (4227 Series; PICO Technology, St. Neots, Cambridgeshire, UK) with the first running at a sampling frequency of 250 MHz and the second at 1 MHz. The time-resolution of the detection system, limited by the detector/preamplifier system response time, was determined to be 15 ns (5). A quantity of Biophysical Journal 109(2) 287-297 50,000 pretrigger points were recorded (corresponding to 200 ms and 50 ms, respectively) before the exciting laser flash and were stored as reference intensity.…”

Section: Time-resolved Ir Spectroscopymentioning

confidence: 99%

“…Other groups stated that E90 deprotonates early in the photocycle and plays a direct role in the gating mechanism, a conclusion mostly supported by molecular dynamics simulations (30). While it is evident that electrostatics drive structural changes in proteins and that helix hydration facilitates ion conductance (5,12,30), a general consensus on how the observed proton transfer steps are linked to channel opening and closure, to our knowledge, has not yet been reached.…”

Section: Introductionmentioning

confidence: 96%

Kinetic and Vibrational Isotope Effects of Proton Transfer Reactions in Channelrhodopsin-2

Resler

Schultz

Lórenz-Fonfrı́a

et al. 2015

Biophysical Journal

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Channelrhodopsins (ChRs) are light-gated cation channels. After blue-light excitation, the protein undergoes a photocycle with different intermediates. Here, we have recorded transient absorbance changes of ChR2 from Chlamydomonas reinhardtii in the visible and infrared regions with nanosecond time resolution, the latter being accomplished using tunable quantum cascade lasers. Because proton transfer reactions play a key role in channel gating, we determined vibrational as well as kinetic isotope effects (VIEs and KIEs) of carboxylic groups of various key aspartic and glutamic acid residues by monitoring their C=O stretching vibrations in H2O and in D2O. D156 exhibits a substantial KIE (>2) in its deprotonation and reprotonation, which substantiates its role as the internal proton donor to the retinal Schiff base. The unusual VIE of D156, upshifted from 1736 cm(-1) to 1738 cm(-1) in D2O, was scrutinized by studying the D156E variant. The C=O stretch of E156 shifted down by 8 cm(-1) in D2O, providing evidence for the accessibility of the carboxylic group. The C=O stretching band of E90 exhibits a VIE of 9 cm(-1) and a KIE of ∼2 for the de- and the reprotonation reactions during the lifetime of the late desensitized state. The KIE of 1 determined in the time range from 20 ns to 5 ms is incompatible with early deprotonation of E90.

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Pre-Gating Conformational Changes in the ChETA Variant of Channelrhodopsin-2 Monitored by Nanosecond IR Spectroscopy

Cited by 39 publications

References 82 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

Temporal evolution of helix hydration in a light-gated ion channel correlates with ion conductance

Kinetic and Vibrational Isotope Effects of Proton Transfer Reactions in Channelrhodopsin-2

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