Directed Evolution of Far-Red Fluorescent Rhodopsins

Microbial rhodopsins are a diverse group of photoactive trans-membrane 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. At least some mutations discovered in one rhodopsin (GR) can also be transferred to another (Arch) to increase fluorescence. In several 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 also 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.