Directed evolution of a far-red fluorescent rhodopsin

作者：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^*

来源：Proceedings of the National Academy of Sciences, 2014, 111(36): 13034-13039.

DOI：10.1073/pnas.1413987111

• optogenetics
• opsins
• bioelectricity
• voltage sensor
• near-infrared

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 fora fluorescent protein (maximal excitation/emission at similar to 620 nm/730 nm).

• 出版日期2014-9-9