title: Directed evolution of a far-red fluorescent rhodopsin.
abstract: 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 pK(a) 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).
authors.affiliation: Divisions of Chemistry and Chemical Engineering and.
authors.forename: R Scott
authors.initials: RS
authors.lastname: McIsaac
authors.affiliation: Divisions of Chemistry and Chemical Engineering and.
authors.forename: Martin K M
authors.initials: MK
authors.lastname: Engqvist
authors.affiliation: Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, 91125; and.
authors.forename: Timothy
authors.initials: T
authors.lastname: Wannier
authors.affiliation: Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, 91125; and.
authors.forename: Adam Z
authors.initials: AZ
authors.lastname: Rosenthal
authors.affiliation: Divisions of Chemistry and Chemical Engineering and.
authors.forename: Lukas
authors.initials: L
authors.lastname: Herwig
authors.affiliation: Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, 91125; and.
authors.forename: Nicholas C
authors.initials: NC
authors.lastname: Flytzanis
authors.affiliation: Department of Physiology and Biophysics, University of California, Irvine School of Medicine, Irvine, CA 92697.
authors.forename: Eleonora S
authors.initials: ES
authors.lastname: Imasheva
authors.affiliation: Department of Physiology and Biophysics, University of California, Irvine School of Medicine, Irvine, CA 92697.
authors.forename: Janos K
authors.initials: JK
authors.lastname: Lanyi
authors.affiliation: Department of Physiology and Biophysics, University of California, Irvine School of Medicine, Irvine, CA 92697.
authors.forename: Sergei P
authors.initials: SP
authors.lastname: Balashov
authors.affiliation: Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, 91125; and.
authors.forename: Viviana
authors.initials: V
authors.lastname: Gradinaru
authors.affiliation: Divisions of Chemistry and Chemical Engineering and Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, 91125; and frances@cheme.caltech.edu.
authors.forename: Frances H
authors.initials: FH
authors.lastname: Arnold
mesh_terms: D019843:Archaeal Proteins / Q000378:metabolism*; D001665:Binding Sites; D002470:Cell Survival; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000378:metabolism; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D055352:Halorubrum / Q000378:metabolism; D006801:Humans; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation; D040681:Structural Homology, Protein; D017494:Proton Pumps
chemical_list: D019843:Archaeal Proteins; D049452:Green Fluorescent Proteins; D050505:Mutant Proteins; C055476:archaerhodopsin protein, Archaea; D017494:Proton Pumps; C485184:enhanced green fluorescent protein
keywords: bioelectricity; near-infrared; opsins; optogenetics; voltage sensor