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

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 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.

Retinal chromophore charge delocalization and confinement explain the extreme photophysics of Neorhodopsin

“…Given the current uncertainty about the regiochemistry of the photoisomerization reaction in NeoR 34 , 35 , we also computed three additional MEPs (see Supplementary Section 5 ) corresponding to the counterclockwise (CCW) isomerization of the C11=C12 and C7=C8 double bond and to the CW isomerization of the C9=C10 double bond. The choice of the CW/CCW pattern for adjacent double bonds conforms to the well-known aborted bicycle-pedal motion 30 , 36 , the archetypal space-saving reaction coordinate for the rPSB chromophore isomerization.…”

Voltage imaging with engineered proton-pumping Rhodopsins: Insights from the proton transfer pathway

“…116 Arch3(D95N) accumulates 13cis N-like species under red illumination, and voltage possibly influences the RSB protonation and therefore the equilibrium between M and N. 116 Combining ultravioletvisible (UV-Vis) absorption, fluorescence and FT-Raman spectroscopy, two Arch3 derivatives, QuasAr2 and No-vArch, were found to be able to cycle between O-like and M-like states using 660 nm and 405 nm illumination. 117 The proton donor mutation D106H was thought to be responsible for the accumulation of the M-like state under red light, as lacking a proton donor to RSB inhibits the M  N transition. 117 Figure 6.…”

“…117 The proton donor mutation D106H was thought to be responsible for the accumulation of the M-like state under red light, as lacking a proton donor to RSB inhibits the M  N transition. 117 Figure 6. Key residues identified for the voltage sensing in Arch3 and Archon1 (PDB: 6GUZ).…”

Rhodopsins: An Excitingly Versatile Protein Species for Research, Development and Creative Engineering

“…The fluorescence of these voltage sensors most likely originates in late-stage photo-intermediates ( Maclaurin et al, 2013 ). The introduced mutations may even result in a complex bistable photo-equilibrium between a fluorescent and a non-fluorescent state ( Mei et al, 2021 ; Penzkofer et al, 2021 ). Meanwhile a host of additional voltage sensors have been developed.…”