Protonation states and pH titration in the photocycle of photoactive yellow protein

Photoactive yellow protein (PYP) undergoes a light-driven cycle of color an d protonation states that is part of a mechanism of bacterial phototaxis. T his article concerns functionally important protonation states of PYP and t he interactions that stabilize them, and changes in the protonation state d uring the photocycle. In particular, the chromophore pK(a) is known to be s hifted down so that the chromophore is negatively charged in the ground sta re (dark state) even though it is buried in the protein, while nearby Glu46 has an unusually high pK(a). The photocycle involves changes of one or bot h of these protonation states. Calculations of pK(a) values and protonation states using a semi-macroscopic electrostatic model are presented for the wild-type and three mutants, in both the ground state and the bleached (I-2 ) intermediate state. Calculations allowing multiple I-I-bonding arrangemen ts around the chromophore also have been carried out. In addition, ground-s tate pK(a) values of the chromophore have been measured by UV-visible spect roscopy for the wild-type and the same three mutants. Because of the unusua l protonation states and strong electrostatic interactions, PYP represents a severe test of the ability of theoretical models to yield correct calcula tions of electrostatic interactions in proteins. Good agreement between exp eriment and theory can be obtained for the ground state provided the protei n interior is assumed to have a relatively low dielectric constant, but onl y partial agreement between theory and experiment is obtained for the bleac hed state. We also present a reinterpretation of previously published data on the pi-I-dependence of the recovery of the ground state from the bleache d state. The new analysis implies a pK(a) value of 6.37 fur Glu46 in the bl eached state, which is consistent with other available experimental data, i ncluding data that only became available after this analysis. The new analy sis suggests that signal transduction is modulated by the titration propert ies of the bleached state, which are in turn determined by electrostatic in teractions; Overall, the results of this study provide a quantitative pictu re of the interactions responsible for the unusual protonation states of th e chromophore and Glu46, and of protonation changes upon bleaching.