Directed evolution of a far-red fluorescent rhodopsin

Significance Archaerhodopsin-3 (Arch) is an integral membrane protein that can function as a genetically encoded fluorescent indicator of membrane voltage in neurons. The ability to visualize changes in membrane voltage is of great interest as a readout for neuronal activity. Published variants of this protein, however, are too dim to enable wide-field imaging of cell populations. We used directed evolution to increase the absolute brightness of Arch as a reporter for optogenetics research and live-cell imaging. This study establishes that introducing mutations around the retinal Schiff-base linkage and screening for increased fluorescence is an effective strategy for generating bright rhodopsin variants. At least some mutations discovered in one rhodopsin (Gloeobacter violaceus rhodopsin) can be transferred to another (Arch) to increase fluorescence. Microbial rhodopsins are a diverse group of photoactive transmembrane proteins found in all three domains of life. A member of this protein family, Archaerhodopsin-3 (Arch) of halobacterium Halorubrum sodomense, was recently shown to function as a fluorescent indicator of membrane potential when expressed in mammalian neurons. Arch fluorescence, however, is very dim and is not optimal for applications in live-cell imaging. We used directed evolution to identify mutations that dramatically improve the absolute brightness of Arch, as confirmed biochemically and with live-cell imaging (in Escherichia coli and human embryonic kidney 293 cells). In some fluorescent Arch variants, the pKa of the protonated Schiff-base linkage to retinal is near neutral pH, a useful feature for voltage-sensing applications. These bright Arch variants enable labeling of biological membranes in the far-red/infrared and exhibit the furthest red-shifted fluorescence emission thus far reported for a fluorescent protein (maximal excitation/emission at ∼620 nm/730 nm).