Dynamic Processes in Metalloprotein Complexes

金属蛋白复合物的动态过程

• 批准号: 9808392
• 负责人: Nenad Kostic
• 金额: $ 30万
• 依托单位: Iowa State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-10-01 至 2002-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9808392&HistoricalAwards=false
• 关键词: Dynamic; Processes; Metalloprotein; Complexes

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.

科斯蒂克MCB 9808392A型技术这项研究的长期目标是在分子水平上解释金属蛋白之间电子转移反应的机制，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。这一理解很重要，因为作为电子载体和氧化还原酶的金属蛋白及其络合物对新陈代谢是必不可少的。目的是了解生物反应的基本原理，特别是与作为生理伙伴的蛋白质的反应。重点不是生物(热)反应的动力学，而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调节整个生物反应的动态过程。然而，这些动态过程可能被热反应所掩盖。在更快的光致电子转移中，感兴趣的过程可以从掩蔽过程中解耦出来，并进行定量的研究。这项研究植根于私家侦探S过去的成就，但却开辟了新的方向。最近对绿藻衣藻细胞色素f、质蓝蛋白和细胞色素c6的三维结构的测定，以及伯克利分校和加州大学洛杉矶分校的合作者在分子生物学方面的成功，为这些生物物理和生化研究奠定了基础。该工作计划结合了应用的分子生物学(定点突变)、光诱导氧化还原反应的动力学(激光闪光光解)和光谱学(核磁共振和光学吸收与发射)，以回答以下问题。(1)什么动力学过程可能调节细胞色素f与其生理上等价的氧化剂--胞浆花青素和细胞色素C6的反应？(2)细胞色素f显著的双域结构如何控制该蛋白质与其生理伙伴之间的识别和反应。(3)细胞色素C6在功能上是否含有与酪蛋白83相同的残基？(4)细胞色素C6在与细胞色素f的反应中是否使用其酸性斑块？蛋白质表面的识别斑块将被结构上的非侵入性突变改变。这些突变和离子强度的动力学效应将揭示更多关于蛋白质-蛋白质结合的信息。如果柔性双蛋白复合体从最初的对接构型重排到不同的反应构型，对溶液粘度和温度的影响的定量分析将揭示这种重排的性质及其轨迹。前面提到的一些问题正在辩论中，其他问题则几乎没有被之前的研究人员触及过。问题不是这样的。3和4来自最新的理论分析，所有的答案都将推动进一步的理论工作。这个项目将激发人们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的基本相互作用和转化，例如蛋白质。电子是所有原子的基本成分，也是带负电荷的最小粒子。活细胞中的电子流动是所有进行光合作用的有机体的基本过程。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。本研究研究了三种蛋白质：细胞色素f、胞质蓝素和细胞色素c6的电子路径的基本步骤，它们都来自相同的绿藻，在这些蛋白质的自然和突变形式中，也有轻微的改变形式。这些蛋白质之间传递电子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由它们的三维结构和蛋白质表面的性质决定，以及蛋白质中有目的的变化如何影响传输速度。现有的理论预测将得到检验，其结果将被用来激励理论家进行新的分析。化学和分子生物学、实验和理论之间的这种相互作用，对研究生命的基本原理是有利的。