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Role of a Conserved Salt Bridge between the PAS Core and the N-Terminal Domain in the Activation of the Photoreceptor Photoactive Yellow Protein

Daniel Hoersch ^*, Harald Otto ^*, Chandra P. Joshi ^*, Berthold Borucki ^*, Michael A. Cusanovich  and Maarten P. Heyn ^*

^* Biophysics Group, Department of Physics, Freie Universität Berlin, Berlin, Germany; and Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, Arizona

Correspondence: Address reprint requests to Maarten P. Heyn, Biophysics Group, Dept. of Physics, Freie Universität Berlin, Arnimallee 14, 14195 Berlin, Germany. Tel.: 49-30-83856160; Fax: 49-30-83856299; E-mail: heyn{at}physik.fu-berlin.de.

The effect of ionic strength on the conformational equilibrium between the I₂ intermediate and the signaling state I₂' of the photoreceptor PYP and on the rate of recovery to the dark state were investigated by time-resolved absorption and fluorescence spectroscopy. With increasing salt concentration up to 600 mM, the recovery rate k₃ decreases and the I₂/I₂' equilibrium (K) shifts in the direction of I₂'. At higher ionic strength both effects reverse. Experiments with mono-(KCl, NaBr) and divalent (MgCl₂, MgSO₄) salts show that the low salt effect depends on the ionic strength and not on the cation or anion species. These observations can be described over the entire ionic strength range by considering the activity coefficients of an interdomain salt bridge. At low ionic strength the activity coefficient decreases due to counterion screening whereas at high ionic strength binding of water by the salt leads to an increase in the activity coefficient. From the initial slopes of the plots of log k₃ and log K versus the square root of the ionic strength, the product of the charges of the interacting groups was found to be –1.3 ± 0.2, suggesting a monovalent ion pair. The conserved salt bridge K110/E12 connecting the ß-sheet of the PAS core and the N-terminal domain is a prime candidate for this ion pair. To test this hypothesis, the mutants K110A and E12A were prepared. In K110A the salt dependence of the I₂/I₂' equilibrium was eliminated and of the recovery rate was greatly reduced below 600 mM. Moreover, at low salt the recovery rate was six times slower than in wild-type. In E12A significant salt dependence remained, which is attributed to the formation of a novel salt bridge between K110 and E9. At high salt reversal occurs in both mutants suggesting that salting out stabilizes the more compact I₂ structure. However, chaotropic anions like SCN shift the I₂/I₂' equilibrium toward the partially unfolded I₂' form. The salt linkage K110/E12 stabilizes the photoreceptor in the inactive state in the dark and is broken in the light-induced formation of the signaling state, allowing the N-terminal domain to detach from the ß-scaffold PAS core.