Absorption and Emission Spectroscopic Investigation of the Thermal Dynamics of the Archaerhodopsin 3 Based Fluorescent Voltage Sensor QuasAr1

Abstract

QuasAr1 is a fluorescent voltage sensor derived from Archaerhodopsin 3 (Arch) of Halorubrum sodomense by directed evolution. Here we report absorption and emission spectroscopic studies of QuasAr1 in Tris buffer at pH 8. Absorption cross-section spectra, fluorescence quantum distributions, fluorescence quantum yields, and fluorescence excitation spectra were determined. The thermal stability of QuasAr1 was studied by long-time attenuation coefficient measurements at room temperature (23 ± 2 °C) and at 2.5 ± 0.5 °C. The apparent melting temperature was determined by stepwise sample heating up and cooling down (obtained apparent melting temperature: 65 ± 3 °C). In the protein melting process the originally present protonated retinal Schiff base (PRSB) with absorption maximum at 580 nm converted to de-protonated retinal Schiff base (RSB) with absorption maximum at 380 nm. Long-time storage of QuasAr1 at temperatures around 2.5 °C and around 23 °C caused gradual protonated retinal Schiff base isomer changes to other isomer conformations, de-protonation to retinal Schiff base isomers, and apoprotein structure changes showing up in ultraviolet absorption increase. Reaction coordinate schemes are presented for the thermal protonated retinal Schiff base isomerizations and deprotonations in parallel with the dynamic apoprotein restructurings.

Photocycle Dynamics of the Archaerhodopsin 3 Based Fluorescent Voltage Sensor QuasAr1

“…The slow reformation of Ret_580 I and Ret_580 II was competing with thermal apoprotein restructuring leading to RSB stabilization without reprotonation. The thermal dynamics of QuasAr1 was studied in a separate paper (apparent melting temperature determination, thermal activated ground-state protonated retinal Schiff base isomerization, deprotonation, and apoprotein restructuring) [33].…”

“…The triple-dotted curve belonging to t exc = 0 (named Ret_580 (t exc = 0)) shows the initial absorption coefficient spectrum of QuasAr1 deprived from retinal isomer contributions other than Ret_580 (dashed triple dotted curve named Residuals). The curves Ret_580 (t exc = 0) and Residuals were determined in [33]. The inset in Figure 1a shows the temporal development of the absorption coefficient α a (t exc ) at the probe wavelength λ pr = 620 nm (long-wavelength absorption region of Ret_580).…”

“…It indicates an initially fast absorption decrease (photoconversion of Ret_580I component) followed by a slow absorption decrease (photoconversion of Ret_580II component). [33]). The dashed triple-dotted curve named Residuals shows the absorption coefficient contribution of residual retinal isomers in QuasAr1 other than Ret_580 (taken from Figure 1 in [33]).…”

“…[33]). The dashed triple-dotted curve named Residuals shows the absorption coefficient contribution of residual retinal isomers in QuasAr1 other than Ret_580 (taken from Figure 1 in [33]). The curve gLED 590 nm (λ) = SLED 590 nm (λ)/SLED 590 nm (λmax) shows the spectral distribution of the excitation light source LED 590 nm.…”

“…The data points are fitted by More detailed information on the photoinduced retinal isomerization and deprotonation dynamics of Ret_580 during light exposure was obtained by subtracting the remaining Ret_580 absorption coefficient contributions αa,Ret_580(λ,texc) at time texc and the initial residual retinal isomers Figure 1 in [33]). The dashed triple-dotted curve named Residuals shows the absorption coefficient contribution of residual retinal isomers in QuasAr1 other than Ret_580 (taken from Figure 1 in [33]). The curve g LED 590 nm (λ) = S LED 590 nm (λ)/S LED 590 nm (λ max ) shows the spectral distribution of the excitation light source LED 590 nm.…”