Directed Evolution of Gloeobacter violaceus Rhodopsin Spectral Properties Martin K.M. Engqvist 1 , R. Scott McIsaac 1 , Peter Dollinger 1 , Nicholas C. Flytzanis 2 , Michael Abrams 1 , Stanford Schor 1 and Frances H. Arnold 1,2 1 - Division of Chemistry and Chemical Engineering, California Institute of Technology, Mail Code 210-41, Pasadena, CA 91125, USA 2 - Division of Biology and Biological Engineering, California Institute of Technology, Mail Code 156-29, Pasadena, CA 91125, USA Correspondence to Frances H. Arnold: Division of Chemistry and Chemical Engineering, California Institute of Technology, Mail Code 210-41, Pasadena, CA 91125, USA. frances@cheme.caltech.edu http://dx.doi.org/10.1016/j.jmb.2014.06.015 Edited by R. Gaudet 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. © 2014 Elsevier Ltd. All rights reserved. Introduction Many proteins, including transcription factors [1,2], enzymes [3–5], and ion pumps/channels [6– 8], have activities that are directly coupled to light. One functionally diverse class of light-activated proteins—opsins—occurs throughout the microbial world, where they facilitate phototactic and photo- phobic responses in algae [9,10], enhanced surviv- al in response to nutrient starvation in bacteria [11], and conversion of solar energy into electrochemical energy in diverse microbes [12,13]. Knowing the molecular determinants for these proteins' light absorption properties will help us to understand their biological functions and to engineer new versions for applications in bioenergy [13], bioma- terials [14], optogenetics [15], and live-cell imaging [16]. Opsins are integral membrane proteins that bind a retinal chromophore to form a colored holoprotein known as rhodopsin. All microbial rhodopsins have similar overall topologies with seven transmembrane helices that enclose all-trans retinal [17,18], which is bound through a covalent Schiff base linkage to the ε-amino group of a conserved lysine located at helix seven. Despite similar structures, rhodopsins show significant variation in the wavelengths of light they absorb, mediated by interactions between retinal and the opsin apoprotein [19]. Naturally occurring microbial rhodopsins exhibit a range of spectral properties, with peak absorption wavelengths (λ max ) between 437 nm and 590 nm [20,21]. In proteorhodopsins (PRs), which IMF YJMBI-64489; No. of pages: 16; 4C: 3, 4, 6, 7, 8 0022-2836/© 2014 Elsevier Ltd. All rights reserved. J. Mol. Biol. (2014) xx, xxx–xxx Article Please cite this article as: Engqvist Martin K.M., et al, Directed Evolution of Gloeobacter violaceus Rhodopsin Spectral Properties, J Mol Biol (2014), http://dx.doi.org/10.1016/j.jmb.2014.06.015