Analog Retinal Redshifts Visible Absorption of QuasAr Transmembrane Voltage Sensors into Near‐infrared

Mei, Gaoxiang; Mamaeva, Natalia; Ganapathy, Srividya; Wang, Peng; DeGrip, Willem J.; Rothschild, Kenneth J.

doi:10.1111/php.13169

Cited by 6 publications

(23 citation statements)

References 69 publications

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“…As previously reported, QuasAr2 exhibits broad excitation/emission peaks with wavelength maxima near 600 and 725 nm, respectively (see figure S3 of (24)). An emission/excitation contour plot of NovArch recorded immediately after 5 min of 405 nm LED illumination to increase the fraction of the O-like species in the sample reveals similar characteristics, with a broad excitation band near 590 nm and red-shifted emission maximum near 725 nm (Fig.…”

Section: Quarar2 Novarch and Ar3-d95q Nir Fluorescencesupporting

confidence: 81%

“…In a proteoliposome environment, the M-like intermediate of QuasAr2 generated by 660 nm illumination also decayed slowly, but with a slightly faster decay constant (85 min, data not shown). In contrast, as previously reported, dark adaptation of QuasAr2 over a 12-hour period did not result in any appreciable decay of the O-like absorbing species (24).…”

Section: Optical Switching Of Quasar2 Between O-like and M-like States And Formation Of A Stable O-like Dark Statesupporting

confidence: 58%

“…Expression, purification and reconstitution of QuasAr2 and NovArch. Methods for the expression of QuasAr2 and NovArch in E. coli cell cultures, purification and reconstitution using His-tag Nickel-chelated nitrilotriacetic acid (Ni-NTA) agarose beads affinity chromatography and reconstitution into in E. coli polar lipid membrane vesicles or detergent n-dodecyl-b-D-maltoside (DDM) micelles were previously reported (24). Briefly, pET 28b(+) vectors encoding QuasAr2 and NovArch genes with a C-terminal His-tag were synthesized by GenScript (Piscataway, NJ).…”

Section: Methodsmentioning

confidence: 99%

“…Fourier transform Raman spectroscopy. FT-Raman measurements of reconstituted QuasAr2 and NovArch in ECPL (proteolipid membrane vesicles) were measured in aqueous buffer at pH 7.3 in glass capillaries as described previously (24). FT-Raman measurements were made using 1064-nm laser excitation on a Bruker MultiRam FT-Raman spectrometer operating at 4 cm À1 resolution and power ranging from 30 to 300 mW.…”

Section: Methodsmentioning

confidence: 99%

“…In order to better understand the molecular basis for some of these limitations and in particular to address limitations 2 and 4 listed above, we used a combination of UV-Vis absorption, FT-Raman and fluorescence spectroscopy to characterize the two closely related OTVSs QuasAr2 and NovArch. We already addressed limitations 1 and 3 in a previous study (24). QuasAr2 has already been used as an OTVS to perform all-optical electrophysiology in mammalian neurons (19).…”

Section: Introductionmentioning

confidence: 99%

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Optical Switching Between Long‐lived States of Opsin Transmembrane Voltage Sensors

Mei

Cavini

Mamaeva

et al. 2021

Photochem & Photobiology

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Opsin-based transmembrane voltage sensors (OTVSs) are membrane proteins increasingly used in optogenetic applications to measure voltage changes across cellular membranes. In order to better understand the photophysical properties of OTVSs, we used a combination of UV-Vis absorption, fluorescence and FT-Raman spectroscopy to characterize Qua-sAr2 and NovArch, two closely related mutants derived from the proton pump archaerhodopsin-3 (AR3). We find both QuasAr2 and NovArch can be optically cycled repeatedly between O-like and M-like states using 5-min exposure to red (660 nm) and near-UV (405 nm) light. Longer red-light exposure resulted in the formation of a long-lived photoproduct similar to pink membrane, previously found to be a photoproduct of the BR O intermediate with a 9-cis retinylidene chromophore configuration. However, unlike QuasAr2 whose O-like state is stable in the dark, NovArch exhibits an O-like state which slowly partially decays in the dark to a stable Mlike form with a deprotonated Schiff base and a 13-cis,15-anti retinylidene chromophore configuration. These results reveal a previously unknown complexity in the photochemistry of OTVSs including the ability to optically switch between different long-lived states. The possible molecular basis of these newly discovered properties along with potential optogenetic and biotechnological applications are discussed.

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Section: Quarar2 Novarch and Ar3-d95q Nir Fluorescencesupporting

confidence: 81%

Section: Optical Switching Of Quasar2 Between O-like and M-like States And Formation Of A Stable O-like Dark Statesupporting

confidence: 58%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Optical Switching Between Long‐lived States of Opsin Transmembrane Voltage Sensors

Mei

Cavini

Mamaeva

et al. 2021

Photochem & Photobiology

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Merocyanines form bacteriorhodopsins with strongly bathochromic absorption maxima

Mackintosh,

Hoischen,

Martin

et al. 2023

Photochem Photobiol Sci

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There is a need to shift the absorbance of biomolecules to the optical transparency window of tissue for applications in optogenetics and photo-pharmacology. There are a few strategies to achieve the so-called red shift of the absorption maxima. Herein, a series of 11 merocyanine dyes were synthesized and employed as chromophores in place of retinal in bacteriorhodopsin (bR) to achieve a bathochromic shift of the absorption maxima relative to bR’s $${\lambda }_{\mathrm{max}}^{a}$$ λ max a of 568 nm. Assembly with the apoprotein bacterioopsin (bO) led to stable, covalently bound chromoproteins with strongly bathochromic absorbance bands, except for three compounds. Maximal red shifts were observed for molecules 9, 2, and 8 in bR where the $${\lambda }_{\mathrm{max}}^{a}$$ λ max a was 766, 755, and 736 nm, respectively. While these three merocyanines have different end groups, they share a similar structural feature, namely, a methyl group which is located at the retinal equivalent position 13 of the polyene chain. The absorption and fluorescence data are also presented for the retinal derivatives in their aldehyde, Schiff base (SB), and protonated SB (PSB) forms in solution. According to their hemicyanine character, the PSBs and their analogue bRs exhibited fluorescence quantum yields (Φf) several orders of magnitude greater than native bR (Φf 0.02 to 0.18 versus 1.5 × 10–5 in bR) while also exhibiting much smaller Stokes shifts than bR (400 to 1000 cm−1 versus 4030 cm−1 in bR). The experimental results are complemented by quantum chemical calculations where excellent agreement between the experimental $${\lambda }_{\mathrm{max}}^{a}$$ λ max a and the calculated $${\lambda }_{\mathrm{max}}^{a}$$ λ max a was achieved with the second-order algebraic-diagrammatic construction [ADC(2)] method. In addition, quantum mechanics/molecular mechanics (QM/MM) calculations were employed to shed light on the origin of the bathochromic shift of merocyanine 2 in bR compared with native bR. Graphical abstract

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Theoretical investigation of the photocycle dynamics of the Archaerhodopsin 3 based fluorescent voltage sensor Archon2

Penzkofer

Silapetere²,

Hegemann³

2023

Journal of Photochemistry and Photobiology A: Chemistry

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Analog Retinal Redshifts Visible Absorption of QuasAr Transmembrane Voltage Sensors into Near‐infrared

Cited by 6 publications

References 69 publications

Optical Switching Between Long‐lived States of Opsin Transmembrane Voltage Sensors

Optical Switching Between Long‐lived States of Opsin Transmembrane Voltage Sensors

Merocyanines form bacteriorhodopsins with strongly bathochromic absorption maxima

Theoretical investigation of the photocycle dynamics of the Archaerhodopsin 3 based fluorescent voltage sensor Archon2

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