title: Directed Evolution of a Bright Near-Infrared Fluorescent Rhodopsin Using a Synthetic Chromophore.
abstract: By engineering a microbial rhodopsin, Archaerhodopsin-3 (Arch), to bind a synthetic chromophore, merocyanine retinal, in place of the natural chromophore all-trans-retinal (ATR), we generated a protein with exceptionally bright and unprecedentedly red-shifted near-infrared (NIR) fluorescence. We show that chromophore substitution generates a fluorescent Arch complex with a 200-nm bathochromic excitation shift relative to ATR-bound wild-type Arch and an emission maximum at 772 nm. Directed evolution of this complex produced variants with pH-sensitive NIR fluorescence and molecular brightness 8.5-fold greater than the brightest ATR-bound Arch variant. The resulting proteins are well suited to bacterial imaging; expression and stability have not been optimized for mammalian cell imaging. By targeting both the protein and its chromophore, we overcome inherent challenges associated with engineering bright NIR fluorescence into Archaerhodopsin. This work demonstrates an efficient strategy for engineering non-natural, tailored properties into microbial opsins, properties relevant for imaging and interrogating biological systems.
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125, USA.
authors.forename: Lukas
authors.initials: L
authors.lastname: Herwig
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125, USA.
authors.forename: Austin J
authors.initials: AJ
authors.lastname: Rice
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125, USA; Division of Biology and Biological Engineering, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125, USA.
authors.forename: Claire N
authors.initials: CN
authors.lastname: Bedbrook
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125, USA.
authors.forename: Ruijie K
authors.initials: RK
authors.lastname: Zhang
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125, USA.
authors.forename: Antti
authors.initials: A
authors.lastname: Lignell
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125, USA.
authors.forename: Jackson K B
authors.initials: JKB
authors.lastname: Cahn
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125, USA.
authors.forename: Hans
authors.initials: H
authors.lastname: Renata
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125, USA.
authors.forename: Sheel C
authors.initials: SC
authors.lastname: Dodani
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125, USA.
authors.forename: Inha
authors.initials: I
authors.lastname: Cho
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125, USA.
authors.forename: Long
authors.initials: L
authors.lastname: Cai
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125, USA.
authors.forename: Viviana
authors.initials: V
authors.lastname: Gradinaru
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125, USA; Division of Biology and Biological Engineering, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125, USA. Electronic address: frances@cheme.caltech.edu.
authors.forename: Frances H
authors.initials: FH
authors.lastname: Arnold
mesh_terms: D001665:Binding Sites; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007536:Isomerism; D007700:Kinetics; D008856:Microscopy, Fluorescence; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D012172:Retinaldehyde / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019265:Spectroscopy, Near-Infrared
chemical_list: D012243:Rhodopsin; D012172:Retinaldehyde
keywords: Archaerhodopsin; directed evolution; live-cell imaging; near-infrared fluorescence; protein engineering; synthetic chromophore substitution