Proton Transfers in a Channelrhodopsin-1 Studied by Fourier Transform Infrared (FTIR) Difference Spectroscopy and Site-directed Mutagenesis

Ogren, John I.; Yi, Adrian; Mamaev, Sergey; Li, Hai; Spudich, John L.; Rothschild, Kenneth J.

doi:10.1074/jbc.m114.634840

Cited by 21 publications

(27 citation statements)

References 44 publications

(83 reference statements)

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“…Resonance Raman spectroscopy data are not consistent with this residue acting as a Schiff base counterion at neutral pH, but it cannot be excluded that Glu68 deprotonates early in the photocycle and accepts a proton from the Schiff base during formation of the M intermediate (149). A similar “two-step” process has been shown by resonance Raman and FTIR-difference spectroscopy for the Asp85 homolog in the cation channelrhodopsin Ca ChR1 (98). The role of Glu68 as a proton acceptor in Gt ACR1 is supported by the Glu68-dependence of an outward proton transfer current evident in a mutant in which the second photoactive site carboxylate, Asp234, is neurtralized (136).…”

Section: The Known Molecular Functions Of Microbial Rhodopsinssupporting

confidence: 59%

“…A novel two-step proton relay mechanism that transfers a proton from the Asp85 homolog to the Asp212 homolog during the primary phototransition and from the Schiff base to the Asp85 homolog during M formation has been proposed for Ca ChR1 based on FTIR (Fourier Transform Infrared) difference spectroscopy (98). Intramolecular proton transfer currents are not detected by patch clamp recording from Cr ChR2 and other high-efficiency CCRs, although an outward intramolecular proton transfer is observed in weaker CCRs such as Ca ChR1 (96).…”

Section: The Known Molecular Functions Of Microbial Rhodopsinsmentioning

confidence: 99%

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Microbial Rhodopsins: Diversity, Mechanisms, and Optogenetic Applications

Govorunova

Sineshchekov

et al. 2017

Annu. Rev. Biochem.

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302

306

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Microbial rhodopsins are a family of photoactive retinylidene proteins widespread throughout the microbial world. They are notable for their diversity of function, using variations of a shared seven-transmembrane helix design and similar photochemical reactions to carry out distinctly different light-driven energy and sensory transduction processes. Their study has contributed to our understanding of how evolution modifies protein scaffolds to create new protein chemistry, and their use as tools to control membrane potential with light is fundamental to the transformative technology of optogenetics. We review the currently known functions, and present more in-depth assessment of three functionally and structurally distinct types discovered over the past two years: (i) anion-conducting channelrhodopsins (ACRs) from cryptophyte algae, enabling efficient optogenetic neural suppression, (ii) cryptophyte cation-conducting channelrhodopsins (CCRs), structurally distinct from the green algae CCRs used extensively for neural activation, and (iii) enzymerhodopsins, with light-gated guanylylcyclase or kinase activity promising for optogenetic control of signal transduction.

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Section: The Known Molecular Functions Of Microbial Rhodopsinssupporting

confidence: 59%

Section: The Known Molecular Functions Of Microbial Rhodopsinsmentioning

confidence: 99%

Microbial Rhodopsins: Diversity, Mechanisms, and Optogenetic Applications

Govorunova

Sineshchekov

et al. 2017

Annu. Rev. Biochem.

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302

306

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“…Steady-state Fourier-transform infrared (FTIR) spectroscopic experiments helped to identify similar conformational changes and (de-) protonation events in ChR2 (6,19,20) and ChR1-ChR2 chimeras (21,22) from C. reinhardtii. Different conformational and protonation changes have been deduced from steady-state FTIR experiments on ChR1 from Chlamydomonas augustae (23)(24)(25)(26). For ChR2 we have further shown by time-resolved FTIR spectroscopy that D253 acts as primary acceptor of the Schiff-base proton and D156 as primary donor (7).…”

Section: Introductionmentioning

confidence: 98%

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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“…Comparison with static FTIR difference spectrum recorded at −23°C revealed that the backbone structural changes at lower temperature are partially blocked. This method has been used routinely in our laboratory to study a wide-range of microbial rhodopsins including sensory rhodopsins [31,32], proteorhodopsins [33,129] and channelrhodopsins [175].…”

Section: Kj Rothschild / the Early Development And Application Of Fmentioning

confidence: 99%

“…These studies were all conducted in collaboration with John Spudich, a leader in the microbial rhodopsin field at the University of Texas Health Science Center at Houston. They included FTIR difference studies along with RRS on halorhodopsin (HR) [197], sensory rhodopsins (SRI, SRII) from archaebacterial [29,31,34,38,184], fungal neurospora rhodopsin (NO) [30], anabaena sensory rhodopsin from cyanobacteria (ASR) [32], green and blue proteorhodopsin proton pumps from marine bacteria (PRs) [4,5,24,28,33,129,130]; archaerhodopsins-3 (AR3), a BR-like protein [56,216] and most recently channelrhodopsins (ChRs) from algae [173,174,176,250]. These proteins span a range of functions which are representative of diverse biomembranes process and include active anion transport (HRs), signal transduction (SRI, SRII and ASR), and light-gated ion channels (ChRs).…”

Section: Beyond Bacteriorhodopsinmentioning

confidence: 99%

The early development and application of FTIR difference spectroscopy to membrane proteins: A personal perspective

Rothschild

2016

BSI

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Abstract. Membrane proteins facilitate some of the most important cellular processes including energy conversion, ion transport and signal transduction. While conventional infrared absorption provides information about membrane protein secondary structure, a major challenge is to develop a dynamic picture of the functioning of membrane proteins at the molecular level. The introduction of FTIR difference spectroscopy around 1980 to study structural changes in membrane proteins along with a number of associated techniques including protein isotope labeling, site-directed mutagenesis, polarization dichroism, attenuated total reflection and time-resolved spectroscopy have led to significant progress towards this goal. It is now possible to routinely detect conformational changes of individual amino acid residues, backbone peptides, binding ligands, chromophores and even internal water molecules under physiological conditions with time-resolution down to nanoseconds. The advent of ultrafast pulsed-IR lasers has pushed this time-resolution down to femtoseconds. The early development of FTIR difference spectroscopy as applied to membrane proteins with special focus on bacteriorhodopsin is reviewed from a personal perspective.

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Proton Transfers in a Channelrhodopsin-1 Studied by Fourier Transform Infrared (FTIR) Difference Spectroscopy and Site-directed Mutagenesis

Cited by 21 publications

References 44 publications

Microbial Rhodopsins: Diversity, Mechanisms, and Optogenetic Applications

Microbial Rhodopsins: Diversity, Mechanisms, and Optogenetic Applications

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

The early development and application of FTIR difference spectroscopy to membrane proteins: A personal perspective

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