Borucki Berthold, Kyndt John A, Joshi Chandra P, Otto Harald, Meyer Terry E, Cusanovich Michael A, Heyn Maarten P
Biophysics Group, Department of Physics, Freie Universität Berlin, Arnimallee 14, 14195 Berlin, Germany.
Biochemistry. 2005 Oct 25;44(42):13650-63. doi: 10.1021/bi050991z.
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'.
我们研究了嗜盐嗜盐红螺菌光活性黄色蛋白（PYP）的突变体Y98Q和Y98F的光循环。Y98位于β4-β5环中，被认为与α3-α4环中的R52相互作用，从而稳定该区域。Y98在所有已知的PYP物种中都是保守的，除了在Ppr和Ppd中它被F取代。我们发现用F取代Y98对光循环动力学没有显著影响。然而，Y98Q突变体观察到了主要变化。我们的结果表明98位需要一个芳香环，特别是对于恢复和正常的I1/I2平衡。Y98的环可以稳定β4-β5环。或者，Y98环可以与异构化的发色团环瞬时相互作用，从而在I1/I2平衡中稳定I2中间体。对于Y98Q，信号状态I2'的衰减减慢了约40倍，I2和I2'中间体的上升减慢了2-3倍。此外，I1中间体与I2/I2'处于pH依赖的平衡状态，在pH 7和50 mM KCl时，I1和I2群体的比例接近1。从pH 5.5到8，平衡向I1移动，pKa约为6.3。在pH 8以上，I1和I2/I2'的群体减少，这是由于I1与另一种在约425 nm处吸收的物种I1'之间的平衡（pKa约为9.8），我们认为I1'是一种具有表面暴露的去质子化发色团的I2样形式。发现I1/I2/I2'平衡强烈依赖于KCl浓度，盐在高达600 mM KCl时稳定信号状态I2'。对这种盐诱导的向I2'的转变进行了分析，并解释为离子与特定位点的结合。此外，通过对振幅光谱的分析，我们得出结论，KCl对I2到I2'的转变起主要作用，即导致信号状态I2'和疏水表面斑块暴露的全局构象变化。在野生型和Y98F中，与Y98Q相比，I1/I2平衡更偏向I2/I2'，但在pH 7时也依赖于盐。I2到I2'的转变似乎由离子锁控制，可能涉及β支架上的K110和N端帽上的E12之间的盐桥。盐结合会破坏盐桥并削弱两个结构域之间的相互作用，促进在形成I2'时N端结构域从β支架上释放。
