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Directed evolution of a far-red fluorescent rhodopsin.
McIsaac, R Scott; Engqvist, Martin K M; Wannier, Timothy; Rosenthal, Adam Z; Herwig, Lukas; Flytzanis, Nicholas C; Imasheva, Eleonora S; Lanyi, Janos K; Balashov, Sergei P; Gradinaru, Viviana; Arnold, Frances H.
Afiliación
  • McIsaac RS; Divisions of Chemistry and Chemical Engineering and.
  • Engqvist MK; Divisions of Chemistry and Chemical Engineering and.
  • Wannier T; Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, 91125; and.
  • Rosenthal AZ; Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, 91125; and.
  • Herwig L; Divisions of Chemistry and Chemical Engineering and.
  • Flytzanis NC; Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, 91125; and.
  • Imasheva ES; Department of Physiology and Biophysics, University of California, Irvine School of Medicine, Irvine, CA 92697.
  • Lanyi JK; Department of Physiology and Biophysics, University of California, Irvine School of Medicine, Irvine, CA 92697.
  • Balashov SP; Department of Physiology and Biophysics, University of California, Irvine School of Medicine, Irvine, CA 92697.
  • Gradinaru V; Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, 91125; and.
  • Arnold FH; Divisions of Chemistry and Chemical Engineering and Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, 91125; and frances@cheme.caltech.edu.
Proc Natl Acad Sci U S A ; 111(36): 13034-9, 2014 Sep 09.
Article en En | MEDLINE | ID: mdl-25157169
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).
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Evolución Molecular Dirigida / Proteínas Arqueales Límite: Humans Idioma: En Revista: Proc Natl Acad Sci U S A Año: 2014 Tipo del documento: Article Pais de publicación: Estados Unidos
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Evolución Molecular Dirigida / Proteínas Arqueales Límite: Humans Idioma: En Revista: Proc Natl Acad Sci U S A Año: 2014 Tipo del documento: Article Pais de publicación: Estados Unidos