Effect of Hofmeister cosolutes on the photocycle of photoactive yellow protein at moderately alkaline pH.

The photocycle of photoactive yellow protein was studied by kinetic absorption spectroscopy from below 100ns to seconds, at moderately alkaline pH, in the presence of high concentrations of various salts. Chemometric analysis combined with multiexponential fit of the flash-induced difference spectra provided evidence for five intermediates, including a spectrally silent form before the final recovery of the parent state, but only three with significantly distinct spectra. The calculated intermediate spectra constituted the input for the following spectrotemporal model fit using a sufficiently complex photocycle scheme with reversible transitions. This yielded the rate coefficients of the molecular transitions, the final spectra and the kinetics of the intermediates. Except for the transition between the two red shifted (early) intermediates (pR1 and pR2) and the final photocycle step, all reactions appeared to be reversible. Kosmotropic and chaotropic cosolutes had a systematic effect on the molecular rate coefficients. The largest effect, associated presumably with the exposure of the hydrophobic interior of the protein, accompanies the transition between the second red-shifted and the first blue-shifted intermediate (pR2 and pB1, respectively), i.e. it coincides with the chromophore protonation. The dependence of the rate coefficients on the Hofmeister cosolutes suggests that the conformational change of photoactive yellow protein leading eventually to the most unfolded signaling state takes place in several steps, and starts already with the relaxation after the chromophore isomerization in the microsecond time domain.