Published in
National Academy of Sciences, Proceedings of the National Academy of Sciences, 36(111), p. 13034-13039, 2014
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- National Academy of Sciences
- ORCID
- [www.ncbi.nlm.nih.gov] | PDF
- [publications.lib.chalmers.se]
- [authors.library.caltech.edu] | PDF
- [cloudfront.escholarship.org] | PDF
- [authors.library.caltech.edu] | PDF
- [europepmc.org] | PDF
- [www.researchgate.net] | PDF
- [www.ncbi.nlm.nih.gov] | PDF
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Directed evolution of a far-red fluorescent rhodopsin
,
Adam Z. Rosenthal,
Lukas Herwig,
Nicholas C. Flytzanis,
Eleonora S. Imasheva,
Janos K. Lanyi,
Sergei P. Balashov,
Viviana Gradinaru,
Frances H. Arnold
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Abstract
Microbial rhodopsins are a diverse group of photoactive transmembrane proteins found in all three domains of life. A member of this protein family, Archaerhodopsin-3 (Arch) of halobacterium Halorubrum sodomense, was recently shown to function as a fluorescent indicator of membrane potential when expressed in mammalian neurons. Arch fluorescence, however, is very dim and is not optimal for applications in live-cell imaging. We used directed evolution to identify mutations that dramatically improve the absolute brightness of Arch, as confirmed biochemically and with live-cell imaging (in Escherichia coli and human embryonic kidney 293 cells). In some fluorescent Arch variants, the pKa of the protonated Schiff-base linkage to retinal is near neutral pH, a useful feature for voltage-sensing applications. These bright Arch variants enable labeling of biological membranes in the far-red/infrared and exhibit the furthest red-shifted fluorescence emission thus far reported for a fluorescent protein (maximal excitation/emission at ∼620 nm/730 nm).