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Directed evolution of a far-red fluorescent rhodopsin

McIsaac, R. Scott and Engqvist, Martin K. M. and Wannier, Timothy and Rosenthal, Adam Z. and Herwig, Lukas and Flytzanis, Nicholas C. and Imasheva, Eleonora S. and Lanyi, Janos K. and Balashov, Sergei P. and Gradinaru, Viviana and Arnold, Frances H. (2014) Directed evolution of a far-red fluorescent rhodopsin. Proceedings of the National Academy of Sciences of the United States of America, 111 (36). pp. 13034-13039. ISSN 0027-8424. PMCID PMC4246972. doi:10.1073/pnas.1413987111.

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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 pK_a 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).

Item Type:Article
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URLURL TypeDescription Information CentralArticle
McIsaac, R. Scott0000-0002-5339-6032
Rosenthal, Adam Z.0000-0002-6936-3665
Flytzanis, Nicholas C.0000-0002-7921-9392
Gradinaru, Viviana0000-0001-5868-348X
Arnold, Frances H.0000-0002-4027-364X
Additional Information:Copyright © 2014 National Academy of Sciences. Contributed by Frances H. Arnold, July 23, 2014 (sent for review June 19, 2014). Published online before print August 25, 2014, doi: 10.1073/pnas.1413987111. This work was funded by the Institute for Collaborative Biotechnologies through Grant W911NF-09-0001 from the US Army Research Office (to F.H.A.); National Institutes of Health (NIH) Grant 1R21MH103824-01 (to F.H.A. and V.G.); NIH Grant 1R01DA028299 (Massachusetts Institute of Technology sub-award 5710002669 to F.H.A.); NIH/National Institute of Neurological Disorders and Stroke New Innovator Award IDP20D017782-01 (to V.G.); NIH Grant GM29498 (to J.K.L. and S.P.B.); Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences, Department of Energy Grant DEFG03-86ER13525 (to J.K.L. and S.P.B.); the German Research Foundation (M.K.M.E.) under Program EN 957/1-1; and California Institute of Technology (Caltech) Biology Division Training Grant NIH/NRSA 5T32GM07616 (to N.C.F.). V.G. acknowledges startup funds from the President and Provost of Caltech, the Biology and Biological Engineering Division of Caltech, and the Beckman Institute of Caltech. R.S.M. acknowledges financial support from the Shurl and Kay Curci Foundation and the Life Sciences Research Foundation. Author contributions: R.S.M., M.K.M.E., and F.H.A. designed research; R.S.M., M.K.M.E., T.W., A.Z.R., L.H., N.C.F., E.S.I., and S.P.B. performed research; R.S.M., M.K.M.E., T.W., L.H., N.C.F., V.G., and F.H.A. contributed new reagents/analytic tools; R.S.M., M.K.M.E., T.W., A.Z.R., N.C.F., E.S.I., J.K.L., S.P.B., and F.H.A. analyzed data; and R.S.M. and F.H.A. wrote the paper. The authors declare no conflict of interest. This article contains supporting information online at
Funding AgencyGrant Number
Army Research Office (ARO)W911NF-09-0001
Department of Energy (DOE)DEFG03-86ER13525
Deutsche Forschungsgemeinschaft (DFG)EN 957/1-1
NIH Predoctoral Fellowship5T32GM07616
President and Provost of CaltechUNSPECIFIED
Caltech Biology and Biological Engineering DivisionUNSPECIFIED
Caltech Beckman InstituteUNSPECIFIED
Shurl and Kay Curci FoundationUNSPECIFIED
Life Sciences Research FoundationUNSPECIFIED
Subject Keywords:optogenetics; opsins; bioelectricity; voltage sensor; near-infrared
Issue or Number:36
PubMed Central ID:PMC4246972
Record Number:CaltechAUTHORS:20140825-213535516
Persistent URL:
Official Citation:R. Scott McIsaac, Martin K. M. Engqvist, Timothy Wannier, Adam Z. Rosenthal, Lukas Herwig, Nicholas C. Flytzanis, Eleonora S. Imasheva, Janos K. Lanyi, Sergei P. Balashov, Viviana Gradinaru, and Frances H. Arnold Directed evolution of a far-red fluorescent rhodopsin PNAS 2014 111 (36) 13034-13039; published ahead of print August 25, 2014, doi:10.1073/pnas.1413987111
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:48877
Deposited By: George Porter
Deposited On:26 Aug 2014 14:39
Last Modified:10 Nov 2021 18:37

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