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.