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Directed evolution of a far-red fluorescent rhodopsin.


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).

SUBMITTER: McIsaac RS 

PROVIDER: S-EPMC4246972 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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Directed evolution of a far-red fluorescent rhodopsin.

McIsaac R Scott RS   Engqvist Martin K M MK   Wannier Timothy T   Rosenthal Adam Z AZ   Herwig Lukas L   Flytzanis Nicholas C NC   Imasheva Eleonora S ES   Lanyi Janos K JK   Balashov Sergei P SP   Gradinaru Viviana V   Arnold Frances H FH  

Proceedings of the National Academy of Sciences of the United States of America 20140825 36


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 improv  ...[more]

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