Demchuk E, Genick U K, Woo T T, Getzoff E D, Bashford D
Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
Biochemistry. 2000 Feb 8;39(5):1100-13. doi: 10.1021/bi991513p.
Photoactive yellow protein (PYP) undergoes a light-driven cycle of color and protonation states that is part of a mechanism of bacterial phototaxis. This article concerns functionally important protonation states of PYP and the interactions that stabilize them, and changes in the protonation state during the photocycle. In particular, the chromophore pK(a) is known to be shifted down so that the chromophore is negatively charged in the ground state (dark state) even though it is buried in the protein, while nearby Glu46 has an unusually high pK(a). The photocycle involves changes of one or both of these protonation states. Calculations of pK(a) values and protonation states using a semi-macroscopic electrostatic model are presented for the wild-type and three mutants, in both the ground state and the bleached (I(2)) intermediate state. Calculations allowing multiple H-bonding arrangements around the chromophore also have been carried out. In addition, ground-state pK(a) values of the chromophore have been measured by UV-visible spectroscopy for the wild-type and the same three mutants. Because of the unusual protonation states and strong electrostatic interactions, PYP represents a severe test of the ability of theoretical models to yield correct calculations of electrostatic interactions in proteins. Good agreement between experiment and theory can be obtained for the ground state provided the protein interior is assumed to have a relatively low dielectric constant, but only partial agreement between theory and experiment is obtained for the bleached state. We also present a reinterpretation of previously published data on the pH-dependence of the recovery of the ground state from the bleached state. The new analysis implies a pK(a) value of 6.37 for Glu46 in the bleached state, which is consistent with other available experimental data, including data that only became available after this analysis. The new analysis suggests that signal transduction is modulated by the titration properties of the bleached state, which are in turn determined by electrostatic interactions. Overall, the results of this study provide a quantitative picture of the interactions responsible for the unusual protonation states of the chromophore and Glu46, and of protonation changes upon bleaching.
光活性黄色蛋白（PYP）经历颜色和质子化状态的光驱动循环，这是细菌趋光性机制的一部分。本文关注PYP功能上重要的质子化状态以及稳定这些状态的相互作用，还有光循环过程中质子化状态的变化。特别地，已知发色团的pK(a)值会下移，使得发色团即使埋在蛋白质中在基态（暗态）时也带负电荷，而附近的Glu46具有异常高的pK(a)值。光循环涉及这两种质子化状态中一种或两种的变化。本文给出了使用半宏观静电模型对野生型和三个突变体在基态和漂白（I(2)）中间态的pK(a)值和质子化状态的计算。还进行了允许发色团周围有多种氢键排列的计算。此外，通过紫外可见光谱法测量了野生型和相同三个突变体发色团的基态pK(a)值。由于存在异常的质子化状态和强烈的静电相互作用，PYP对理论模型在蛋白质中产生正确静电相互作用计算能力构成了严峻考验。如果假设蛋白质内部具有相对较低的介电常数，对于基态实验和理论之间可以获得良好的一致性，但对于漂白态理论和实验之间仅获得部分一致性。我们还对先前发表的关于从漂白态恢复基态的pH依赖性数据进行了重新解释。新的分析表明，在漂白态下Glu46的pK(a)值为6.37，这与其他现有实验数据一致，包括在此分析之后才获得的数据。新的分析表明，信号转导由漂白态的滴定性质调节，而漂白态的滴定性质又由静电相互作用决定。总体而言，本研究结果提供了一幅关于导致发色团和Glu46异常质子化状态的相互作用以及漂白时质子化变化的定量图景。
