Abstract
The retinal photocycle dynamics of the fluorescent voltage sensor QuasAr1 (Archaerhodopsin 3 P60S-T80S-D95H-D106H-F161V mutant from Halorubrum sodomense) in pH 8 Tris buffer was studied. The samples were photoexcited to the first absorption band of the protonated retinal Schiff base (PRSB) Ret_580 (absorption maximum at λmax ≈ 580 nm), and the retinal Schiff base photoisomerization and protonation state changes were followed by absorption spectra recordings during light exposure and after light exposure. Ret_580 turned out to be composed of two protonated retinal Schiff base isomers, namely Ret_580I and Ret_580II. Photoexcitation of Ret_580I resulted in barrier-involved isomerization to Ret_540 (quantum yield ≈ 0.056) and subsequent retinal proton release leading to Ret_410 deprotonated retinal Schiff base (RSB). In the dark, Ret_410 partially recovered to Ret_580I and partially stabilized to irreversible Ret_400 due to apoprotein restructuring (Ret_410 lifetime ≈ 2 h). Photoexcitation of Ret_580II resulted in barrier-involved isomerization to Ret_640 (quantum yield ≈ 0.00135) and subsequent deprotonation to Ret_370 (RSB). In the dark, Ret_370 partially recovered to Ret_580II and partially stabilized to irreversible Ret_350 due to apoprotein restructuring (Ret_370 lifetime ≈ 10 h). Photocycle schemes and reaction coordinate diagrams for Ret_580I and Ret_580II were developed and photocyle parameters were determined.
Highlights
Tracking membrane potential of cells, especially neurons, using fluorescence methods is of high interest and is an active field of research [1,2,3,4,5,6,7,8,9,10]
Most microbial rhodopsin voltage indicators are based on Archaerhodopsin 3 (Arch) from Halorubrum sodomense [29] and variants thereof obtained by mutations (Arch D95N [29], Arch D95N-D106E [27], Arch D95Q-D106E [30], Archer1 (=Arch D95E-T99C) [31], Archer2 (=Arch D95E-T99C-A225M) [31], QuasAr1 (=Arch P60S-T80S-D95H-D106H-F161V) [26], QuasAr2 (=QuasAr1 H95Q) [26], QuasAr3 (=QuasAr2 K171R) [28], paQuasAr3 (=QuasAr3 V59A) [28], Archon1 (=Arch T20S-G41A-V44E-P60S-T80P-D86N-D95Q-D106H-A136T-F161V-T183I-L197I-G241Q) [32], and Archon2 (=Arch T56P-P60S-T80P-D95H-T99S-T116I-F161V-T183I-L197I-A225C) [32])
The QuasAr1 samples in pH 8 Tris buffer were photoexcited to the first absorption band in the green-yellow-orange spectral range, and the retinal photoisomerization and protonation state changes were followed by absorption spectra recordings during light exposure and after light exposure
Summary
Tracking membrane potential of cells, especially neurons, using fluorescence methods is of high interest and is an active field of research (change of membrane voltage causes change of fluorescence efficiency) [1,2,3,4,5,6,7,8,9,10]. Most microbial rhodopsin voltage indicators are based on Archaerhodopsin 3 (Arch) from Halorubrum sodomense [29] and variants thereof obtained by mutations (Arch D95N [29], Arch D95N-D106E [27], Arch D95Q-D106E [30], Archer (=Arch D95E-T99C) [31], Archer (=Arch D95E-T99C-A225M) [31], QuasAr1 (=Arch P60S-T80S-D95H-D106H-F161V) [26], QuasAr2 (=QuasAr1 H95Q) [26], QuasAr3 (=QuasAr2 K171R) [28], paQuasAr3 (=QuasAr3 V59A) [28], Archon (=Arch T20S-G41A-V44E-P60S-T80P-D86N-D95Q-D106H-A136T-F161V-T183I-L197I-G241Q) [32], and Archon (=Arch T56P-P60S-T80P-D95H-T99S-T116I-F161V-T183I-L197I-A225C) [32]). The mutations improved the fluorescence intensity dependence on membrane voltage and the membrane localization [26,28,31,32]
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