Directed evolution of gloeobacter violaceus rhodopsin spectral properties
Journal article, 2015

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.

opsins

optogenetics

proton pumps

fluorescent proteins

Retinal

Author

Martin Engqvist

R.S. McIsaac

P. Dollinger

N.C. Flytzanis

M. Abrams

S. Schor

F.H. Arnold

Journal of Molecular Biology

0022-2836 (ISSN)

Vol. 427 1 205-220

Subject Categories

Biochemistry and Molecular Biology

DOI

10.1016/j.jmb.2014.06.015

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Created

10/10/2017