Optical Switching Between Long‐lived States of Opsin Transmembrane Voltage Sensors. (14th May 2021) - Mei, Gaoxiang
Abstract: 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 partially decays 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. Abstract : Opsin‐based transmembrane voltage sensors (OTVSs) are NIR fluorescence emitting membrane proteins increasingly used in optogenetic applications to measure voltage changes across cellular membranes for instance in brain imaging studies. A combination of UV‐Vis absorption, fluorescence and FT‐Raman spectroscopy was used to characterize QuasAr2 and NovArch, two closely related OTVSs derived from the proton pump archaerhodopsin‐3. Both QuasAr2 and NovArch can be optically switched repeatedly between long‐lived O‐like and M‐like states revealing a previously unknown complexity in the photochemistry of these OTVSs. ​