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Directed evolution of a far-red fluorescent rhodopsin.

  • Author(s): McIsaac, R Scott
  • Engqvist, Martin KM
  • Wannier, Timothy
  • Rosenthal, Adam Z
  • Herwig, Lukas
  • Flytzanis, Nicholas C
  • Imasheva, Eleonora S
  • Lanyi, Janos K
  • Balashov, Sergei P
  • Gradinaru, Viviana
  • Arnold, Frances H
  • et al.
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 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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