Protonation and Conformational Control of Photosynthetic Reaction Center Function

9631063 Wraight光合作用反应中心(RC)为研究内部静电学和构象迁移率在蛋白质功能中的作用提供了一个几乎理想的系统，本项目集中在两个重要主题上：(1)蛋白质内质子转移，它在几乎所有的催化作用中都起着关键作用；(2)对内部电荷产生和置换做出反应的构象弛豫，在催化功能中也具有普遍的重要性。针对RB的RCS，提出了具体的实验方案。Sphaeroides和RPS。绿色，两者都有已知的原子结构。(I)对细菌RC的L、M和H亚基中选定的残基进行定点突变，以探讨它们在质子转移中的作用以及QA和QB的还原。电荷移动将通过RC颜料上的电致变色效应、膜系统上的直接电测量和红外光谱来监测(II)渗透压和溶剂同位素效应的研究，以探索内部水作为蛋白质内质子储存库的作用，以及散装水在QB位的苯醌和苯二酚交换中的作用。这两种影响都将通过测量RC在高光强下的多次周转来进行测试，以揭示稳态周转中的限制步骤。(Iii)研究P+QA-电荷复合作为温度和预光照的函数，使用电荷复合动力学作为能量的分析，并将颜料的光谱响应作为内部电场的分析。这项研究将通过离子环境和pH来控制RC基态，并通过选择突变体来探索重要构象事件的性质。这项工作将在它工作的温度范围内扩展到P+QB-复合。蛋白质结构和功能的所有方面都依赖于分子内原子和基团之间无数的静电相互作用。蛋白质功能的几乎所有方面都需要分子的运动，从单个原子到大分子各部分的协调运动。这样的运动响应并产生静电效应，使两者紧密地交织在一起，近年来得到了越来越多的认识。尽管具有非常普遍的重要性，但适合研究这些过程的实验系统极其有限，特别是对于动态事件，它们基本上局限于光激活的蛋白质反应。该项目利用细菌的光合作用反应中心(RC)作为研究这些基本重要过程的理想模型系统。RC的光驱动反应在蛋白质内部产生电荷运动，引发分子运动(构象松弛)，然后改变电荷的后续行为。通过诱变选择性地改变RC的结构，并通过冷却到低温来限制RC状态的时间演化，可以解除这些事件的纠缠。作为蛋白质功能循环的一部分，构象运动也是允许小分子进出蛋白质所必需的。这也将从质子如何通过蛋白质到达它们的反应部位，蛋白质内部的水如何参与这一过程，以及当其他分子进入和离开它们的活性部位时水如何来去等方面进行研究。***

9631063 Wraight The photosynthetic reaction center (RC) provides an almost ideal system for studying the roles of internal electrostatics and conformational mobility in protein function, and this project focusses on two important topics: (1) intra-protein proton transfer, which plays a crucial role in almost all catalysis, and (2) conformational relaxations in response to internal charge generation and displacement, also of universal importance in catalytic function. Specific experiments are proposed for RCs from Rb. sphaeroides and Rps. viridis, both of which have known atomic structures. (i) Site-specific mutagenesis of selected residues in the L, M and H subunits of the bacterial RC, chosen to probe their role in proton transfer coupled to the reduction of QA and QB. Charge movements will be monitored by electrochromic effects on the RC pigments, by direct electrical measurements on membrane systems, and by infra- red spectroscopy (ii) Studies of osmotic pressure and solvent isotope effects to probe the role of internal waters as a reservoir of protons inside the protein, and of bulk water in the exchange of quinone and quinol at the QB site. Both effects will be assayed by measurements of multiple turnovers of the RC, driven at high light intensity, to reveal limiting steps in the steady-state turnover. (iii) Studies of P+ QA - charge recombination as a function of temperature and pre-illumination, using the charge recombination kinetics as an assay of the energentics, and spectral responses of the pigments as an assay of the internal electric field. The research will probe the nature of the important conformation events by controlling the RC ground state through the ionic milieu and the pH, and by using selected mutants. This work will be extented to P+ QB- recombination, over the temperature range that it works. %%% All aspects of protein structure and function depend on the myriad of electrostatic interactions between atoms and groups inside the molecule. Almost all aspects of protein function require movement of the molecule, at all levels of organization from individual atoms to coordinated motions of sections of the macromolecule. Such movements respond to and generate electrostatic effects, so that the two are intimately entwined., have been increasingly recognized in recent years. Although of very general importance, experimental systems suitable for studying these processes are extremely limited and, especially with respect to dynamic events, they are essentially restricted to light activated protein reactions. This project utilizes the photosynthetic reaction center (RC) from a bacterium as an ideal model system for studying these fundamentally important processes. The light-driven reaction of the RC generates charge movements inside the protein, which initiate molecular motions (conformational relaxations) which then modify the subsequent behavior of the charges. These events can be disentangled by selective modification of the structure of the RC by mutagenesis, and by restricting the time-evolution of the RC states by cooling to low temperatures. Conformational motions are also necessary to allow small molecules in and out of a protein, as part of the protein's functional cycle. This, too, will be studied in terms of how protons pass through the protein to reach their site of reaction, how water inside the protein is involved in this, and how water comes and goes as other molecules enter and leave their active sites. ***