Mechanism of voltage-sensitive fluorescence in a microbial rhodopsin

Maclaurin, Dougal; Venkatachalam, Veena; Lee, Hohjai; Cohen, Adam E.

doi:10.1073/pnas.1215595110

Cited by 139 publications

(201 citation statements)

References 36 publications

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“…Because the A225M mutation did not further increase fluorescence in Arch(DETC), we chose to focus on Arch(DETC) for further characterization and engineering. Although an initial mechanistic study of voltage-sensitive fluorescence in Arch was recently reported (18), it remained unclear which amino acid residues should be targeted for mutation to improve fluorescent properties [other than the Schiff-base counter ion, which is known to affect the lifetime of the fluorescence excited state in bacteriorhodopsin (16) and is already mutated in Arch(DETC)]. To identify mutations that increase Arch(DETC) fluorescence, we thus followed an unbiased approach by performing random mutagenesis over the whole protein (details in Materials and Methods) and screened 2,640 mutants in a 96-well plate assay at neutral pH ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…A recent spectroscopic analysis suggested that fluorescence of wild-type Arch is low but can be elevated through a sequential three-photon process where two consecutive quanta initialize formation of a highly fluorescent state Q formed from the photocycle intermediate N (18). An alternative approach is utilization of mutations of the Schiff-base counter ion (15) to increase the fluorescence yield in the initial (unphotolyzed) state.…”

Section: Discussionmentioning

confidence: 99%

“…Site-saturation libraries were generated using a modified version of the QuikChange method (Stratagene), as previously described (15). For the single-site-saturation libraries, a mix of three mutagenic primers containing the triplet sequences NDT, VHG, and TGG were used in ratio of 12:9:1 for mutagenic PCR amplification (Table S1, primers [7][8][9][10][11][12][13][14][15][16][17][18][19][20]. For the double-site-saturation library, a mixture of 9 mutagenic primers (and a reverse primer) was used for PCR amplification (Table S1, primers [21][22][23][24][25][26][27][28][29][30].…”

Section: Methodsmentioning

confidence: 99%

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Directed evolution of a far-red fluorescent rhodopsin

McIsaac¹,

Engqvist²,

Wannier

et al. 2014

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

115

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Microbial rhodopsins are a diverse group of photoactive transmembrane proteins found in all three domains of life. A member of this protein family, Archaerhodopsin-3 (Arch) of halobacterium Halorubrum sodomense, was recently shown to function as a fluorescent indicator of membrane potential when expressed in mammalian neurons. Arch fluorescence, however, is very dim and is not optimal for applications in live-cell imaging. We used directed evolution to identify mutations that dramatically improve the absolute brightness of Arch, as confirmed biochemically and with livecell imaging (in Escherichia coli and human embryonic kidney 293 cells). In some fluorescent Arch variants, the pK a of the protonated Schiff-base linkage to retinal is near neutral pH, a useful feature for voltage-sensing applications. These bright Arch variants enable labeling of biological membranes in the far-red/infrared and exhibit the furthest red-shifted fluorescence emission thus far reported for a fluorescent protein (maximal excitation/emission at ∼620 nm/730 nm).optogenetics | opsins | bioelectricity | voltage sensor | near-infrared

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Directed evolution of a far-red fluorescent rhodopsin

McIsaac¹,

Engqvist²,

Wannier

et al. 2014

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

115

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show abstract

“…132 Recently, genetically encoded opsins, such as mutants of Arch, have been adapted as voltage sensors. 133 The Arch-derived sensor Arch(D95H) has been shown to have the fluorescence response variations in both amplitude and time course due to voltage change. 134,135 In addition, the Arch variants Arch-EEN and Arch-EEQ have also been demonstrated as voltage sensors with faster kinetics and larger signal intensities compared with Arch(D95H).…”

Section: 131mentioning

confidence: 99%

Toward microendoscopy-inspired cardiac optogeneticsin vivo: technical overview and perspective

Klimas

Entcheva

2014

J. Biomed. Opt

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Abstract. The ability to perform precise, spatially localized actuation and measurements of electrical activity in the heart is crucial in understanding cardiac electrophysiology and devising new therapeutic solutions for control of cardiac arrhythmias. Current cardiac imaging techniques (i.e. optical mapping) employ voltage-or calciumsensitive fluorescent dyes to visualize the electrical signal propagation through cardiac syncytium in vitro or in situ with very high-spatiotemporal resolution. The extension of optogenetics into the cardiac field, where cardiac tissue is genetically altered to express light-sensitive ion channels allowing electrical activity to be elicited or suppressed in a precise cell-specific way, has opened the possibility for all-optical interrogation of cardiac electrophysiology. In vivo application of cardiac optogenetics faces multiple challenges and necessitates suitable optical systems employing fiber optics to actuate and sense electrical signals. In this technical perspective, we present a compendium of clinically relevant access routes to different parts of the cardiac electrical conduction system based on currently employed catheter imaging systems and determine the quantitative size constraints for endoscopic cardiac optogenetics. We discuss the relevant technical advancements in microendoscopy, cardiac imaging, and optogenetics and outline the strategies for combining them to create a portable, miniaturized fiber-based system for all-optical interrogation of cardiac electrophysiology in vivo.

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“…The fluorescence of wild-type Arch-3 appears not to come from the ground state, but from a photogenerated intermediate (see 1 ). Thus a model would need to adopt the correct retinal isomerization state and opsin conformation to reproduce the fluorescence.…”

mentioning

confidence: 99%

A voltage-dependent fluorescent indicator for optogenetic applications, archaerhodopsin-3: Structure and optical properties from in silico modeling

et al. 2017

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It was demonstrated in recent studies that some rhodopsins can be used in optogenetics as fluorescent indicators of membrane voltage. One of the promising candidates for these applications is archaerhodopsin-3. However, the fluorescent signal for wild-type achaerhodopsin-3 is not strong enough for real applications. Rational design of mutants with an improved signal is an important task, which requires both experimental and theoretical studies. Herein, we used a homology-based computational approach to predict the three-dimensional structure of archaerhodopsin-3, and a Quantum Mechanics/Molecular Mechanics (QM/MM) hybrid approach with high-level multireference ab initio methodology (SORCI+Q/AMBER) to model optical properties of this protein. We demonstrated that this methodology allows for reliable prediction of structure and spectral properties of archaerhodopsin-3. The results of this study can be utilized for computational molecular design of efficient fluorescent indicators of membrane voltage for modern optogenetics on the basis of archaerhodopsin-3.

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Mechanism of voltage-sensitive fluorescence in a microbial rhodopsin

Cited by 139 publications

References 36 publications

Directed evolution of a far-red fluorescent rhodopsin

Directed evolution of a far-red fluorescent rhodopsin

Toward microendoscopy-inspired cardiac optogeneticsin vivo: technical overview and perspective

A voltage-dependent fluorescent indicator for optogenetic applications, archaerhodopsin-3: Structure and optical properties from in silico modeling

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