A Caltech Library Service

Directed Evolution of Gloeobacter violaceus Rhodopsin Spectral Properties

Engqvist, Martin K. M. and McIsaac, R. Scott and Dollinger, Peter and Flytzanis, Nicholas C. and Abrams, Michael and Schor, Stanford and Arnold, Frances H. (2015) Directed Evolution of Gloeobacter violaceus Rhodopsin Spectral Properties. Journal of Molecular Biology, 427 (1). pp. 205-220. ISSN 0022-2836. doi:10.1016/j.jmb.2014.06.015.

PDF - Accepted Version
See Usage Policy.


Use this Persistent URL to link to this item:


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 violacaeus 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 incurrred 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-four 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 wild-type protein.

Item Type:Article
Related URLs:
URLURL TypeDescription
McIsaac, R. Scott0000-0002-5339-6032
Flytzanis, Nicholas C.0000-0002-7921-9392
Arnold, Frances H.0000-0002-4027-364X
Additional Information:© 2014 Elsevier B.V. Received date: 28 April 2014; Revised date: 5 June 2014; Accepted date: 22 June 2014. Available online 28 June 2014. We thank Janos Lanyi for kindly providing us with the GR gene used in this project, Naoki Kamo for helpful comments on the proton-pumping assay, and Timothy Wannier for critically reviewing our manuscript and assisting with quantum yield measurements. MKM. E. received funding from the German Research Foundation (DFG) under program EN 957/1-1. Research reported in this publication was supported by the National Institute of Mental Health of the National Institutes of Health (R21MH103824) and by the Institute of Collaborative Biotechnologies (contract W911NF-09-D-0001) through the U.S. Army Research Office. The content is solely the responsibility of the authors and does not represent the official views of any of the funding agencies.
Funding AgencyGrant Number
Deutsche Forschungsgemeinschaft (DFG)EN 957/1-1
Army Research Office (ARO)W911NF-09-D-0001
National Institute of Mental Health (NIMH)UNSPECIFIED
Issue or Number:1
Record Number:CaltechAUTHORS:20140707-083154824
Persistent URL:
Official Citation:Martin K.M. Engqvist, R. Scott McIsaac, Peter Dollinger, Nicholas C. Flytzanis, Michael Abrams, Stanford Schor, Frances H. Arnold, Directed Evolution of Gloeobacter violaceus Rhodopsin Spectral Properties, Journal of Molecular Biology, Volume 427, Issue 1, 16 January 2015, Pages 205-220, ISSN 0022-2836, (
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:46866
Deposited By: Tony Diaz
Deposited On:07 Jul 2014 16:27
Last Modified:10 Nov 2021 17:30

Repository Staff Only: item control page