期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 111, 期 36, 页码 13034-13039出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1413987111
关键词
optogenetics; opsins; bioelectricity; voltage sensor; near-infrared
资金
- US Army Research Office [W911NF-09-0001]
- National Institutes of Health (NIH) [1R21MH103824-01, 1R01DA028299, 5710002669, GM29498]
- NIH/National Institute of Neurological Disorders and Stroke New Innovator Award [IDP20D017782-01]
- Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences, Department of Energy [DEFG03-86ER13525]
- German Research Foundation [EN 957/1-1]
- California Institute of Technology (Caltech) Biology Division Training Grant [NIH/NRSA 5T32GM07616]
- Provost of Caltech
- Biology and Biological Engineering Division of Caltech
- Beckman Institute of Caltech
- Shurl and Kay Curci Foundation
- Life Sciences Research Foundation
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).
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