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 beta4-beta5 loop and is thought to interact with R52 in the alpha3-alpha4 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 I1/I2 equilibrium. The ring of Y98 could stabilize the beta4-beta5 loop. Alternatively, the Y98 ring could transiently interact with the isomerized chromophore ring, thereby stabilizing the I2 intermediate in the I1/I2 equilibrium. For Y98Q, the decay of the signaling state I2' was slowed by a factor of approximately 40, and the rise of the I2 and I2' intermediates was slowed by a factor of 2-3. Moreover, the I1 intermediate is in a pH-dependent equilibrium with I2/I2' with the ratio of the I1 and I2 populations close to one at pH 7 and 50 mM KCl. From pH 5.5 to 8, the equilibrium shifts toward I1, with a pKa of approximately 6.3. Above pH 8, the populations of I1 and I2/I2' decrease due to an equilibrium between I1 and an additional species I1' which absorbs at approximately 425 nm (pKa approximately 9.8) and which we believe to be an I2-like form with a surface-exposed deprotonated chromophore. The I1/I2/I2' equilibrium was found to be strongly dependent on the KCl concentration, with salt stabilizing the signaling state I2' up to 600 mM KCl. This salt-induced transition to I2' 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 I2 to I2' transition, i.e., the global conformational change leading to the signaling state I2' and the exposure of a hydrophobic surface patch. In wild type and Y98F, the I1/I2 equilibrium is more on the side of I2/I2' as compared to Y98Q but is also salt-dependent at pH 7. The I2 to I2' 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 I2'.