Photocycle dynamics of the Archaerhodopsin 3 based fluorescent voltage sensor Archon2.

The retinal photocycle dynamics of the fluorescent voltage sensor Archon2 in pH 8 Tris buffer was studied. Archon2 is a mutant of Archaerhodopsin 3 (Arch) from Halorubrum sodomense obtained by a robotic multidimensional directed evolution approach (Archon2 = Arch T56P-P60S-T80P-D95H-T99S-T116I-F161V-T183I-L197I-A225C). The samples were photo-excited to the first absorption band of the protonated retinal Schiff base (PRSB) Ret_586 (absorption maximum at λ = 586 nm, excitation wavelengths λ = 590 nm and 632.8 nm). The photocycle dynamics were studied by recording absorption spectra during light exposure and after light exposure. Ret_586 photoisomerized to Ret_535 (main component) and Ret_485 (minor component). Ret_535 backward photoisomerized to Ret_586 in light-adapted state (named Ret_586) and partly deprotonated to neutral retinal Schiff base (RSB) Ret_372 in light adapted state (named Ret_372, same isomer form as Ret_535). After excitation light switch-off Ret_372 recovered to Ret_372 in dark-adapted state (Ret_372) which slowly re-protonated to Ret_535, and Ret_535 slowly isomerized back to Ret_586 in dark-adapted state (Ret_586). Photocycle schemes and reaction coordinate diagrams are developed and photocycle parameters are determined.