Directed Evolution of Gloeobacter violaceus Rhodopsin Spectral Properties. Issue 1 (16th January 2015)

• Record Type: Journal Article
• Authors: Engqvist, Martin K.M.; McIsaac, R. Scott; Dollinger, Peter; Flytzanis, Nicholas C.; Abrams, Michael; Schor, Stanford; Arnold, Frances H.
• Is Part Of: Journal of molecular biology. Volume 427:Issue 1(2015:Jan. 01)
• Page Start: 205
• Page End: 220
• Publication Date: 2015-01-16
• Subjects: BR bacteriorhodopsin -- GR Gloeobacter violaceus rhodopsin -- PR proteorhodopsin -- XR xanthorhodopsin -- PPR proton-pumping rhodopsin -- SrSRI Salinibacter ruber sensory rhodopsin I -- DDM n-dodecyl β-d-maltoside -- AUC area under the curve -- Ni-NTA nickel-nitrilotriacetic acid -- NCBI National Center for Biotechnology Information; optogenetics -- retinal -- fluorescent proteins -- opsins -- proton pumps; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2014.06.015
• Languages: English
• ISSNs: 0022-2836

Abstract

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 ± 80 nm, 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 42 nm and 22 nm, 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. Graphical abstract: Highlights: Performed mutagenesis of the retinal-binding pocket of a PPR. Shifts in absorption maxima of up to ± 80 nm were achieved. A subset of mutants exhibited strong fluorescence in the far red/infrared. A first step toward brighter opsin-based biological sensors.