| Autor: |
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. |
| Abstrakt: |
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). |
