Abstract
Archaerhodopsin‐3 (AR3) is a member of the microbial rhodopsin family of hepta‐helical transmembrane proteins, containing a covalently bound molecule of all‐trans retinal as a chromophore. It displays an absorbance band in the visible region of the solar spectrum (λmax 556 nm) and functions as a light‐driven proton pump in the archaeon Halorubrum sodomense. AR3 and its mutants are widely used in neuroscience as optogenetic neural silencers and in particular as fluorescent indicators of transmembrane potential. In this study, we investigated the effect of analogs of the native ligand all‐trans retinal A1 on the spectral properties and proton‐pumping activity of AR3 and its single mutant AR3 (F229S). While, surprisingly, the 3‐methoxyretinal A2 analog did not redshift the absorbance maximum of AR3, the analogs retinal A2 and 3‐methylamino‐16‐nor‐1,2,3,4‐didehydroretinal (MMAR) did generate active redshifted AR3 pigments. The MMAR analog pigments could even be activated by near‐infrared light. Furthermore, the MMAR pigments showed strongly enhanced fluorescence with an emission band in the near‐infrared peaking around 815 nm. We anticipate that the AR3 pigments generated in this study have widespread potential for near‐infrared exploitation as fluorescent voltage‐gated sensors in optogenetics and artificial leafs and as proton pumps in bioenergy‐based applications.
Highlights
Microbial rhodopsins are a family of hepta-helical transmembrane proteins found in a diverse array of micro-organisms spanning archaea, bacteria and eukaryotes
These expression levels are in the same range as achieved with Gloeobacter violaceus rhodopsin (GR) and PR, while enhanced expression was observed for GR (F234S) (GR-FS) relative to GR [22,23]
With the exception of phenyl retinal (PHE), all the retinal analogs tested in this study (Fig. 1) could be stably incorporated in both AR3 and AR3(F229S) and yielded excellent regeneration levels when supplemented in cell culture
Summary
Microbial rhodopsins are a family of hepta-helical transmembrane proteins found in a diverse array of micro-organisms spanning archaea, bacteria and eukaryotes. They function as light-driven ion pumps, channels or sensors and play a vital role in survival and adaptation of their host organisms [1,2]. Light-gated ion channels are expressed in neurons and used to selectively depolarize or hyperpolarize cells upon illumination in the field of optogenetics [7]. This enables precise spatiotemporal control of neuronal activity
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