Figure 1.
Dark recovery kinetics of YtvAL to YtvAD.
A. Dark recovery of YtvAL to YtvAD in E. coli colonies expressing YtvA deposited on a glass coverslip, after 5 minutes illumination with LED465, monitored through fluorescence emission of YtvAD. The blue bars indicate illumination periods with LED465, black bars indicate that colonies were kept in the dark. T = 25°C. B. Dark relaxation of YtvAL to YtvAD, as monitored by the recovery of fluorescence emission after switching off LED465. Blue, buffered YtvA solution; red, E. coli colonies expressing YtvA soaked in 10 mM phosphate buffer solution, containing 0.9% NaCl (W/V), pH = 7.4; green, E. coli colonies expressing YtvA smeared on a glass coverslip. T = 25°C. Black solid lines correspond to fits with a stretched exponential relaxation (E. coli colonies expressing YtvA smeared on a glass coverslip) or with an exponential decay (YtvA solution and bacteria soaked in a buffer). C. Dark relaxation of YtvAL to YtvAD after 5 minutes illumination with LED465 of B. subtilis colonies expressing YtvA. Green curves were measured for colonies smeared on a glass plate, red curves were obtained for colonies soaked in an isotonic buffer. Black curves are the best fits with an exponential decay (colonies soaked in a buffer) or a stretched exponential decay (colonies smeared on a glass plate). Fitting parameters are reported in Table 1.
Table 1.
Apparent time constants for the YtvAL to YtvAD relaxation at 25°C.
Figure 2.
Dark recovery kinetics of dried YtvA.
A. Absorption spectrum for YtvA molecules air-dried on a quartz plate before (black) and after (red) photoconversion with LED465. The green curve reports the absorption spectrum at t = 24660 s at 25°C. After 24 hours the YtvAD spectrum is fully recovered (not shown). B. Dark recovery kinetics of YtvAL to YtvAD followed through the absorbance at 475 nm. The red solid line is the best fit with a double stretched exponential relaxation, with τ = 600±400 s and β = 0.6±0.2 (16%) and τ = (3.7±0.3)×104 s and β = 1.0±0.2 (84%).
Figure 3.
FPALM images of E. coli cells expressing wt YtvA.
FPALM images of E. coli cells expressing wt YtvA soaked in isotonic buffer (A) and under de-hydrated conditions (B). Intracellular distribution seems similar for the system in both hydration states, the protein remains widespread over the cell body with some major aggregates at the cell wall. Experimental conditions for the de-hydrated state: Intensity: 0.2 W/cm2 at 405 nm and 0.05 kW/cm2 at 488 nm; for the hydrated state: Intensity: 1–2 W/cm2 at 405 nm and 0.2 kW/cm2 at 488 nm; frame rate: 30 Hz and total number of collected frames: 20000. Scale bar: 1 µm.
Figure 4.
Dark recovery kinetics of E. coli colonies expressing selected YtvA mutants.
A. Dark relaxation of YtvAL to YtvAD after 5 minutes illumination with LED465 of E. coli colonies expressing the R63K mutant of YtvA. B. Dark relaxation of YtvAL to YtvAD after 5 minutes illumination with LED465 of E. coli colonies expressing Q123N YtvA. Green curves were measured for colonies smeared on a glass coverslip, red curves were obtained for colonies soaked in an isotonic buffer. Black curves are the best fits to stretched exponential decays. Parameters are reported in Table 1.
Figure 5.
Three dimensional arrangement of mutated amino acids in YtvA.
Left. Closeup of the YtvA chromophore (in green) with the amino acids considered in this study represented in capped sticks (red oxygen, blue nitrogen). Right. Solvent accessible surface visualization of the protein with the chromophore in green and the amino acids considered in this study represented in capped sticks.