title: Directed evolution of Gloeobacter violaceus rhodopsin spectral properties.
abstract: Proton-pumping rhodopsins (PPRs) are photoactive retinal-binding proteins that transport ions across biological membranes in response to light. These proteins are interesting for light-harvesting applications in bioenergy production, in optogenetics applications in neuroscience, and as fluorescent sensors of membrane potential. Little is known, however, about how the protein sequence determines the considerable variation in spectral properties of PPRs from different biological niches or how to engineer these properties in a given PPR. Here we report a comprehensive study of amino acid substitutions in the retinal-binding pocket of Gloeobacter violaceus rhodopsin (GR) that tune its spectral properties. Directed evolution generated 70 GR variants with absorption maxima shifted by up to ±80nm, extending the protein's light absorption significantly beyond the range of known natural PPRs. While proton-pumping activity was disrupted in many of the spectrally shifted variants, we identified single tuning mutations that incurred blue and red shifts of 42nm and 22nm, respectively, that did not disrupt proton pumping. Blue-shifting mutations were distributed evenly along the retinal molecule while red-shifting mutations were clustered near the residue K257, which forms a covalent bond with retinal through a Schiff base linkage. Thirty eight of the identified tuning mutations are not found in known microbial rhodopsins. We discovered a subset of red-shifted GRs that exhibit high levels of fluorescence relative to the WT (wild-type) protein.
authors: Division of Chemistry and Chemical Engineering, California Institute of Technology, Mail Code 210-41, Pasadena, CA 91125, USA.
authors: Martin K M
authors: MK
authors: Engqvist
authors: Division of Chemistry and Chemical Engineering, California Institute of Technology, Mail Code 210-41, Pasadena, CA 91125, USA.
authors: R Scott
authors: RS
authors: McIsaac
authors: Division of Chemistry and Chemical Engineering, California Institute of Technology, Mail Code 210-41, Pasadena, CA 91125, USA.
authors: Peter
authors: P
authors: Dollinger
authors: Division of Biology and Biological Engineering, California Institute of Technology, Mail Code 156-29, Pasadena, CA 91125, USA.
authors: Nicholas C
authors: NC
authors: Flytzanis
authors: Division of Chemistry and Chemical Engineering, California Institute of Technology, Mail Code 210-41, Pasadena, CA 91125, USA.
authors: Michael
authors: M
authors: Abrams
authors: Division of Chemistry and Chemical Engineering, California Institute of Technology, Mail Code 210-41, Pasadena, CA 91125, USA.
authors: Stanford
authors: S
authors: Schor
authors: Division of Chemistry and Chemical Engineering, California Institute of Technology, Mail Code 210-41, Pasadena, CA 91125, USA; Division of Biology and Biological Engineering, California Institute of Technology, Mail Code 156-29, Pasadena, CA 91125, USA. Electronic address: frances@cheme.caltech.edu.
authors: Frances H
authors: FH
authors: Arnold
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000458:Cyanobacteria / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D019020:Directed Molecular Evolution; D005453:Fluorescence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D017494:Proton Pumps / Q000378:metabolism*; D025604:Rhodopsins, Microbial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D017494:Proton Pumps; D025604:Rhodopsins, Microbial
keywords: fluorescent proteins; opsins; optogenetics; proton pumps; retinal