Role of a conserved salt bridge between the PAS core and the N-terminal domain in the activation of the photoreceptor photoactive yellow protein |
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| Authors: | Hoersch Daniel Otto Harald Joshi Chandra P Borucki Berthold Cusanovich Michael A Heyn Maarten P |
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| Affiliation: | Biophysics Group, Department of Physics, Freie Universit?t Berlin, Berlin, Germany. |
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| Abstract: | The effect of ionic strength on the conformational equilibrium between the I(2) intermediate and the signaling state I(2)' 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 approximately 600 mM, the recovery rate k(3) decreases and the I(2)/I(2)' equilibrium (K) shifts in the direction of I(2)'. At higher ionic strength both effects reverse. Experiments with mono-(KCl, NaBr) and divalent (MgCl(2), MgSO(4)) 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 k(3) 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 beta-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 I(2)/I(2)' equilibrium was eliminated and of the recovery rate was greatly reduced below approximately 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 I(2) structure. However, chaotropic anions like SCN shift the I(2)/I(2)' equilibrium toward the partially unfolded I(2)' 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 beta-scaffold PAS core. |
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| Keywords: | PAS domain, acronym formed from the names of the first three proteins recognized as sharing this sensory domain PYP, photoactive yellow protein |
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