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：(由申请者提供)本项目重点是创建一个 用于详细研究氧化还原决定因素的模型黄素蛋白 黄素酶的潜在调谐，一个氧化还原蛋白家族，与许多 遗传病。一种使用两种综合迭代重新设计的双边方法 和噬菌体展示定向进化方法将被用来产生 高度稳定、高亲和力的模型黄素蛋白。这个模型蛋白质将允许 深入研究质子和质子的动力学细节的独特机会 黄素蛋白中的电子转移及其偶联。从热力学的角度看 结合的黄素将被电位法研究。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 用快速扫描蛋白质膜伏安法测定pH。参与的氨基酸残基 在还原期间向黄素输送的质子将使用以下方法进行识别 溶液核磁共振。超大天体的定量化整体分析 已发表的黄素蛋白结构和还原潜力 将执行以提供一套工程原则，这些原则将有助于 具有一系列氧化还原特性的黄素蛋白模板的构建。 这些原则将通过纳入特定的 黄素-蛋白质相互作用进入模型黄素蛋白，然后 结合黄素-S氧化还原性质变化的测定。AS 质子转移事件是黄素氧化还原化学的固有部分， 理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潜力的关键一步 调谐黄素蛋白。