Photocycle Dynamics of the Archaerhodopsin 3 Based Fluorescent Voltage Sensor QuasAr1

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 lambda(max) approximate to 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_580(I) and Ret_580(II). Photoexcitation of Ret_580(I) resulted in barrier-involved isomerization to Ret_540 (quantum yield approximate to 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_580(I) and partially stabilized to irreversible Ret_400 due to apoprotein restructuring (Ret_410 lifetime approximate to 2 h). Photoexcitation of Ret_580(II) resulted in barrier-involved isomerization to Ret_640 (quantum yield approximate to 0.00135) and subsequent deprotonation to Ret_370 (RSB). In the dark, Ret_370 partially recovered to Ret_580(II) and partially stabilized to irreversible Ret_350 due to apoprotein restructuring (Ret_370 lifetime approximate to 10 h). Photocycle schemes and reaction coordinate diagrams for Ret_580(I) and Ret_580(II) were developed and photocyle parameters were determined.