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Abstract Opsin‐based transmembrane voltage sensors (OTVSs) are increasingly important tools for neuroscience enabling neural function in complex brain circuits to be explored in live, behaving animals. However, the visible wavelengths required for fluorescence excitation of the current generation of OTVSs limit optogenetic imaging in the brain to depths of only a few mm due to the strong absorption and scattering of visible light by biological tissues. We report that substitution of the native A1 retinal chromophore of the widely used QuasAr1/2 OTVSs with the retinal analog MMAR containing a methylamino‐modified dimethylphenyl ring results in over a 100‐nm redshift of the maxima of the absorption and fluorescence emission bands to near 700 and 840 nm, respectively. FT‐Raman spectroscopy reveals that at pH 7 QuasAr1 with both the A1 and MMAR chromophores possess predominantly an all‐transprotonated Schiff base configuration with the MMAR chromophore exhibiting increased torsion of the polyene single‐/double‐bond system similar to the O‐intermediate of the BR photocycle. In contrast, the A1 and the MMAR chromophores of QuasAr2 exist partially in a 13‐cisPSB configuration. These results demonstrate that QuasArs containing the MMAR chromophore are attractive candidates for use as NIR‐OTVSs, especially for applications such as deep brain imaging.
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Optical Switching Between Long‐lived States of Opsin Transmembrane Voltage Sensors
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 QuasAr2 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 decays partially in the dark to a stable M-like 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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- Award ID(s):
- 1706322
- PAR ID:
- 10226890
- Date Published:
- Journal Name:
- Photochemistry and Photobiology
- ISSN:
- 0031-8655
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1111/php.13428
