Model Protein Studies of Flavin Redox Potential Tuning

黄素氧化还原电位调节的模型蛋白质研究

• 批准号: 6525397
• 负责人: Ronald Koder
• 金额: $ 4.42万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6525397
• 关键词: X ray crystallography; acidity /alkalinity; biochemical evolution; bioenergetics; biotechnology; circular dichroism; computer simulation; cytochrome b; electrochemistry; electron transport; flavoproteins; mathematical model; microcalorimetry; model design /development; nuclear magnetic resonance spectroscopy; oxidation reduction reaction; peptide chemical synthesis; physical model; potentiometry; protein engineering; protein structure function; proteomics; protonation; statistics /biometry; structural biology; thermodynamics

DESCRIPTION: (provided by applicant) This project focuses on the creation of a model flavoprotein for the detailed investigation of the determinants of redox potential tuning in flavoenzymes, a family of redox proteins implicated in many genetic diseases. A bilateral approach using both synthetic iterative redesign and phage display directed evolution methodologies will be used to generate a highly stable, high-affinity model flavoprotein. This model protein will allow the unique opportunity of examining in depth the dynamic details of proton and electron transfers and their coupling in flavoproteins. The thermodynamics of the bound flavin will be investigated potentiometrically. Electron transfer rates and rate-determining proton transfer rates will be measured as a function of pH using fast scan protein film voltametry. Amino acid residues involved in the delivery of protons to the flavin during reduction will be identified using solution NMR. A quantitative global analysis of the extraordinarily large body of flavoprotein structures and reduction potentials which have been published will be performed to provide a set of engineering principles which will aid in the construction of flavoprotein maquettes with a range of redox properties. These principles will be demonstrated by the incorporation of specific flavin-protein interactions into the model flavoprotein followed by the determination of the changes induced in the bound flavin?s redox properties. As proton transfer events are an intrinsic part of flavin redox chemistry, understanding the coupling between electron and proton transfer in these model proteins is a crucial step towards a greater comprehension of redox potential tuning in flavoproteins.

描述：（由申请人提供）该项目的重点是创建一个 用于详细研究氧化还原决定因素的模型黄素蛋白 黄素酶的潜在调节，黄素酶是一个氧化还原蛋白家族，涉及许多 遗传疾病。使用综合迭代重新设计的双边方法 噬菌体展示定向进化方法将用于生成 高度稳定、高亲和力的模型黄素蛋白。该模型蛋白质将允许 深入研究质子和质子动态细节的独特机会 电子转移及其在黄素蛋白中的偶联。热力学 结合的黄素将通过电位分析进行研究。电子转移 速率和决定速率的质子转移速率将作为函数进行测量 使用快速扫描蛋白膜伏安法测定 pH。涉及的氨基酸残基 还原过程中质子向黄素的传递将通过以下方式确定 溶液核磁共振。对异常庞大身体的定量全球分析 已发表的黄素蛋白结构和还原电位 将提供一套工程原理，这将有助于 具有一系列氧化还原特性的黄素蛋白模型的构建。 这些原则将通过结合具体的 黄素-蛋白质相互作用形成模型黄素蛋白，然后 测定结合黄素氧化还原特性引起的变化。作为 质子转移事件是黄素氧化还原化学的固有组成部分， 了解这些模型中电子和质子转移之间的耦合 蛋白质是更好地理解氧化还原电位的关键一步 黄素蛋白的调节。