Effect of salt and pH on the activation of photoactive yellow protein and gateway mutants Y98Q and Y98F

We investigated the photocycle of mutants Y98Q and Y98F of the photoactive yellow protein (PYP) from Halorhodospira halophila. Y98 is located in the beta(4)-beta(5) loop and is thought to interact with R52 in the alpha(3)-alpha(4) loop thereby stabilizing this region. Y98 is conserved in all known PYP species, except in Ppr and Ppd where it is replaced by F. We find that replacement of Y98 by F has no significant effect on the photocycle kinetics. However, major changes were observed with the Y98Q mutant. Our results indicate a requirement for an aromatic ring at position 98, especially for recovery and a normal I-1/I-2 equilibrium. The ring of Y98 could stabilize the beta(4)-beta(5) loop. Alternatively, the Y98 ring could transiently interact with the isomerized chromophore ring, thereby stabilizing the I-2 intermediate in the I-1/I-2 equilibrium. For Y98Q, the decay of the signaling state U was slowed by a factor of similar to 40, and the rise of the I-2 and I-2' intermediates was slowed by a factor of 2-3. Moreover, the I-1 intermediate is in a pH-dependent equilibrium with with the ratio of the I-1 and I-2 populations close to one at pH 7 and 50 mM KCL From pH 5.5 to 8, the equilibrium shifts toward I-1, with a pK(a) of similar to 6.3. Above pH 8, the populations of I-1 and I-2/I-2' decrease due to an equilibrium between I-1 and an additional species I-1' which absorbs at similar to 425 nm (pK(a) similar to 9.8) and which we believe to be an I-2-like form with a surface-exposed deprotonated chromophore. The I-1/I-2/I-2' equilibrium was found to be strongly dependent on the KCl concentration, with salt stabilizing the signaling state I-2(') Up to 600 mM KCl. This salt-induced transition to U was analyzed and interpreted as ion binding to a specific site. Moreover, from analysis of the amplitude spectra, we conclude that KCl exerts its major effect on the I-2 to U transition, i.e., the global conformational change leading to the signaling state I-2(') and the exposure of a hydrophobic surface patch. In wild type and Y98F, the I-1/I-2' equilibrium is more on the side Of I-2/I-2' as compared to Y98Q but is also salt-dependent at pH 7. The I-2 to I-2' transition appears to be controlled by an ionic lock, possibly involving the salt bridge between K110 on the beta-scaffold and E12 on the N-terminal cap. Salt binding would break the salt bridge and weaken the interaction between the two domains, facilitating the release of the N-terminal domain from the beta-scaffold in the formation of I-2'.