Model Protein Studies of Flavin Redox Potential Tuning

描述：（由申请人提供）本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 潜在的调整黄素酶，一个家庭的氧化还原蛋白牵连在许多 遗传病一个双边的方法使用合成迭代重新设计 和噬菌体展示定向进化方法将用于产生 高度稳定、高亲和力的黄素蛋白模型。这种蛋白质模型可以让 深入研究质子动力学细节的独特机会， 黄素蛋白中的电子转移及其偶联。的热力学 结合的黄素将被电位测量法研究。电子转移 速率和速率决定质子转移率将被测量为函数 用快速扫描蛋白膜伏安法测定pH值。参与的氨基酸残基 在还原过程中质子向黄素的传递将用 溶液NMR。超大型天体的全球定量分析 的黄素蛋白结构和还原潜力， 将提供一套工程原理， 具有一系列氧化还原性质的黄素蛋白模型的构建。 这些原则将通过纳入具体的 黄素-蛋白质相互作用转化为模型黄素蛋白， 测定结合黄素引起的变化？的氧化还原性质。作为 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 蛋白质是对氧化还原电位更好理解的关键一步 调节黄素蛋白。

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.