A Schiff base connectivity switch in sensory rhodopsin signaling

Sineshchekov, Oleg A.; Sasaki, Jun; Phillips, Brian J.; Spudich, John L.

doi:10.1073/pnas.0807486105

Cited by 30 publications

(58 citation statements)

References 53 publications

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“…7 Furthermore, as reported previously 7 and shown in Figure 5, the substitution of Ser for His166 inverts the SB deprotonation to the EC side. Therefore, an alternative proton acceptor exists.…”

Section: Discussionsupporting

confidence: 70%

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His166 Is the Schiff Base Proton Acceptor in Attractant Phototaxis Receptor Sensory Rhodopsin I

et al. 2014

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Photoactivation of attractant phototaxis receptor sensory rhodopsin I (SRI) in Halobacterium salinarum entails transfer of a proton from the retinylidene chromophore’s Schiff base (SB) to an unidentified acceptor residue on the cytoplasmic half-channel, in sharp contrast to other microbial rhodopsins, including the closely related repellent phototaxis receptor SRII and the outward proton pump bacteriorhodopsin, in which the SB proton acceptor is an aspartate residue salt-bridged to the SB in the extracellular (EC) half-channel. His166 on the cytoplasmic side of the SB in SRI has been implicated in the SB proton transfer reaction by mutation studies, and mutants of His166 result in an inverted SB proton release to the EC as well as inversion of the protein’s normally attractant phototaxis signal to repellent. Here we found by difference Fourier transform infrared spectroscopy the appearance of Fermi-resonant X–H stretch modes in light-minus-dark difference spectra; their assignment with 15N labeling and site-directed mutagenesis demonstrates that His166 is the SB proton acceptor during the photochemical reaction cycle of the wild-type SRI–HtrI complex.

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Section: Discussionsupporting

confidence: 70%

“…Similar measurements were used to calculate the charge shift ratio of the same mutants by Sineshchekov et al 7 to establish the charge movement inversion and suppression effect confirmed here.…”

Section: Resultssupporting

confidence: 60%

His166 Is the Schiff Base Proton Acceptor in Attractant Phototaxis Receptor Sensory Rhodopsin I

et al. 2014

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“…A similar inversion of protein function was previously observed in haloarchaeal sensory rhodopsin I (SRI), in which a single point mutation either of the photoreceptor itself or of its cognate transducer converted SRI from an attractant to a repellent photoreceptor (16,17). The SRI inversion is attributable to a switch in its conformation from the C (retinylidene Schiff base accessible from the cytoplasm) to E (Schiff base accessible from the extracellular space) conformer (18)(19)(20). The stable open channel of GtACR1_E68R is likely to be particularly valuable for structural comparisons with the WT by enabling identification of residue determinants and structural changes distinguishing the closed and open conformations of the channel by structural methods such as molecular spectroscopy and crystallography.…”

Section: Discussionmentioning

confidence: 99%

Gating mechanisms of a natural anion channelrhodopsin

Sineshchekov

Govorunova

et al. 2015

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

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Anion channelrhodopsins (ACRs) are a class of light-gated channels recently identified in cryptophyte algae that provide unprecedented fast and powerful hyperpolarizing tools for optogenetics. Analysis of photocurrents generated by Guillardia theta ACR 1 (GtACR1) and its mutants in response to laser flashes showed that GtACR1 gating comprises two separate mechanisms with opposite dependencies on the membrane voltage and pH and involving different amino acid residues. The first mechanism, characterized by slow opening and fast closing of the channel, is regulated by Glu-68. Neutralization of this residue (the E68Q mutation) specifically suppressed this first mechanism, but did not eliminate it completely at high pH. Our data indicate the involvement of another, yetunidentified pH-sensitive group X. Introducing a positive charge at the Glu-68 site (the E68R mutation) inverted the channel gating so that it was open in the dark and closed in the light, without altering its ion selectivity. The second mechanism, characterized by fast opening and slow closing of the channel, was not substantially affected by the E68Q mutation, but was controlled by Cys-102. The C102A mutation reduced the rate of channel closing by the second mechanism by ∼100-fold, whereas it had only a twofold effect on the rate of the first. The results show that anion conductance by ACRs has a fundamentally different structural basis than the relatively well studied conductance by cation channelrhodopsins (CCRs), not attributable to simply a modification of the CCR selectivity filter.ion transport | channel gating | channelrhodopsins | optogenetics R ecently we reported a class of rhodopsins from the cryptophyte alga Guillardia theta that act as anion-conducting channels when expressed in cultured animal cells and therefore can be used to suppress neuronal firing by light (1). These proteinsnamed anion channelrhodopsins (ACRs)-show distant sequence homology to cation channelrhodopsins (CCRs) from chlorophyte (green) algae (2), but completely lack permeability for protons and metal cations. This property, as well as large current amplitudes and fast kinetics, makes ACRs superior hyperpolarizing optogenetic tools, compared with proton and chloride pumps (3, 4), or engineered Cl − -conducting CCR variants (5, 6). Two G. theta (Gt) ACRs differ in their spectral sensitivity and channel kinetics (1). GtACR1, with its absorption peak at 515 nm (Fig. S1), has an advantage over a more blue-shifted GtACR2 with maximal efficiency at 470 nm, because it allows using light of longer wavelengths that is less scattered by biological tissue.Because a CCR could be altered to exhibit Cl − channel activity by mutation of a single amino acid residue (5), a fundamental question about ACRs is whether they differ from CCRs only by their selectivity filter. In our search for an answer, we analyzed photocurrents generated by GtACR1 in HEK293 cells under single-turnover conditions in which secondary photochemistry does not complicate the photocycle. We found that GtACR1 cond...

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“…Additionally, as the chemical shift value of the Pintermediate is significantly different from that of the Mintermediate, a rather large conformational change was observed, which may cause large changes in protein interactions, acting as a switch between positive and negative phototaxis functions. [16] After the P-intermediate was generated and trapped stationary under irradiation with 365 nm LED light of the Mintermediate, light illumination was stopped and the accumulation of the P-intermediate under dark conditions was observed (Figure 2 B). The signal intensity of the P-intermediate at 24.8 ppm decreased and that of the G-state at 13.8 ppm increased, indicating that the P-intermediate relaxed to the G-state.…”

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confidence: 99%

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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A Schiff base connectivity switch in sensory rhodopsin signaling

Abstract: Sensory rhodopsin I (SRI) in

Cited by 30 publications

References 53 publications

His166 Is the Schiff Base Proton Acceptor in Attractant Phototaxis Receptor Sensory Rhodopsin I

His166 Is the Schiff Base Proton Acceptor in Attractant Phototaxis Receptor Sensory Rhodopsin I

Gating mechanisms of a natural anion channelrhodopsin

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

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