Dynamic Processes in Metalloprotein Complexes

KosticMCB 9808392A。这项研究的长期目标是在分子水平上解释金属蛋白之间的电子转移反应机制，以及蛋白质-蛋白质对接和动态重排过程对这些反应的重要性。 这种理解是重要的，因为金属蛋白及其复合物，作为电子载体和氧化还原酶，是必不可少的代谢。 其目的是了解生物反应的基本原理，特别是与蛋白质的生理伙伴。 而不是生物（热）反应的动力学，重点是使用密切相关的非生物（光诱导）氧化还原反应的动力学作为一种工具来阐明动态过程，调节整体生物反应。 然而，这些动态过程可能在热反应中被掩盖。 在光诱导电子转移，这是快得多，感兴趣的过程可以从掩蔽过程解耦，并定量研究。这项研究植根于P.I.过去的成就，但在新的方向上取得了成就。 最近确定的三维结构的细胞色素f，plastocyanin，和细胞色素c6的绿色藻类莱茵衣藻和成功的合作者在伯克利分校和加州大学洛杉矶分校的分子生物学为这些生物物理和生物化学的调查。 工作计划结合了应用分子生物学（定点诱变）、光致氧化还原反应动力学（激光闪光光解）和光谱学（NMR和光学吸收和发射），以回答以下问题。(1)什么样的动力学过程可能调节细胞色素f与其生理等效氧化剂质体蓝素和细胞色素c6的反应？(2)细胞色素f的两个结构域是如何控制这种蛋白质与其生理伙伴之间的识别和反应的？(3)细胞色素c6是否含有与质体蓝素中Tyr 83功能相当的残基？(4)细胞色素c6在与细胞色素f的反应中是否使用酸性物质？蛋白质表面的识别补丁将被结构非侵入性突变改变。 这些突变和离子强度的动力学效应将揭示许多关于蛋白质-蛋白质缔合的信息。 如果灵活的双蛋白复合物重新排列从初始的，对接配置到一个不同的，反应性的配置，定量分析的溶液粘度和温度的影响将揭示这种重排的性质和它的轨迹。 上述问题中的一些正在辩论中，其他问题几乎没有被以前的研究人员触及。 问题不。3和4产生于最近的理论分析，所有的答案将刺激进一步的理论工作。 这个项目将激发对生物系统中识别和电子转移的结构-功能关系的思考和重新思考。在最基本的层面上，生命的过程是化学过程。因此，化学的实验和理论方法可以揭示涉及生物分子（如蛋白质）的基本相互作用和转化。电子是所有原子的基本组成部分，也是携带负电荷的最小粒子。电子在活细胞中的流动是进行光合作用的所有生物体的基本过程。因为所有形式的生命都是相互依赖的，细胞中的电子流动是生命的基本要求。细胞中的电子载体是金属蛋白--含有金属原子的蛋白质。 本研究涉及三种蛋白质的电子路径的基本步骤：细胞色素f，质体蓝素，细胞色素c6，都来自同一个绿色植物，在其自然和突变形式的这些蛋白质，也略有改变的形式。这些蛋白质之间电子转移的速度是用激光方法测量的。实验旨在确定这些蛋白质的功能如何受其三维结构和蛋白质表面性质的控制，以及蛋白质中有目的的改变如何影响转移速度。 现有的理论预测将被检验，其结果将用于激励理论家进行新的分析。化学与分子生物学、实验与理论之间的这种相互作用有利于研究生命的基本原理。

KosticMCB 9808392A. technical The long-term goal of this research is to explain, at the molecular level, the mechanism of electron-transfer reactions between metalloproteins and the importance of protein-protein docking and of dynamic rearrangement processes for these reactions. This understanding is important because metalloproteins and their complexes, as electron carriers and redox enzymes, are essential to metabolism. The objective is to understand fundamental principles of biological reactivity, specifically with proteins that are physiological partners. Instead of kinetics of biological (thermal) reactions, the focus is to use kinetics of closely related abiological (photoinduced) redox reactions as a tool to elucidate dynamic processes that modulate the overall biological reactions. These dynamic processes, however, may be masked in the thermal reactions. In the photoinduced electron transfer, which is much faster, the processes of interest can be decoupled from the masking processes and investigated quantitatively. The study is rooted in P.I.'s past accomplishments, but strikes in new directions. Recent determinations of the three-dimensional structures of cytochrome f, plastocyanin, and cytochrome c6 from the green algae Chlamydomonas reinhardtii and successes by collaborators at Berkeley and UCLA in molecular biology set the stage for these biophysical and biochemical investigations. The plan of work combines applied molecular biology (site-directed mutagenesis), kinetics of photoinduced redox reactions (laser flash photolysis), and spectroscopy (NMR and optical absorption and emission) in order to answer the following questions. (1) What dynamic processes possibly modulate the reaction of cytochrome f with its physiologically- equivalent oxidants, plastocyanin and cytochrome c6? (2) How does the remarkable two-domain structure of cytochrome f control the recognition and the reaction between this protein and its physiological partners. (3) Does cytochrome c6 contain a residue functionally equivalent to Tyr 83 in plastocyanin? (4) Does cytochrome c6 use its acidic patch in the reaction with cytochrome f ? Recognition patches on the protein surfaces will be altered by structurally-noninvasive mutations. Kinetic effects of these mutations and of ionic strength will reveal much about the protein-protein association. If flexible diprotein complexes rearrange from the initial, docking configuration to a different, reactive configuration, quantitative analysis of the effects of solution viscosity and of temperature will reveal the nature of this rearrangement and its trajectory. Some of the aforementioned issues are being debated, and others have barely been touched by previous researchers. The questions nos. 3 and 4 arose from very recent theoretical analyses, and all of the answers will spur further theoretical work. This project will stimulate thinking and rethinking about structure-function relationships for recognition and electron transfer in biological systems.2. Non-technicalAt the most elementary level, processes of life are chemicalprocesses. Therefore, experimental and theoretical methods of chemistry can reveal fundamental interactions and transformations involving biological molecules, such as proteins. Electrons are fundamental constituents of all atoms and the smallest particles carrying negative electrical charge. Flow of electrons in the living cell is an essential process for all organisms that carry out photosynthesis. Because all forms of life are interdependent, electron flow in cells is an essential requirement for life. Electron carriers in cells are metalloproteins - proteins containing metal atoms. This study deals with basic steps on the electron paths of three proteins: Cytochrome f, plastocyanin, and cytochrome c6, all from the same green alga, in their natural and mutated forms of these proteins and also with slightly altered forms. The speeds at which these proteins transfer the electrons between themselves are measured by laser methods. Experiments are designed to determine how the function of these proteins is governed by their three-dimensional structures and properties of the protein surfaces, and how purposeful alterations in the proteins affect the speed of transfer. Existing theoretical predictions will be tested and the results used to spur theorists to new analyses. This interplay between chemistry and molecular biology and between experiment and theory is advantageous in the study of fundamental principles of life.