Directed evolution of a far-red fluorescent rhodopsin
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).
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 www.pnas.org/lookup/suppl/doi:10.1073/pnas.1413987111/-/DCSupplemental.
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