STUDIES ON ELECTRON TRANSFER DOMAINS AND COMPLEXES OF FLAVOPROTEINS

黄素蛋白电子传递域和复合物的研究

• 批准号: 6271984
• 负责人: FRANK E FRERMAN
• 金额: $ 18.78万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-05-01 至 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6271984
• 关键词: Escherichia coli; chemical kinetics; circular dichroism; electron transport; flavin adenine dinucleotide; flavoproteins; gene expression; gene mutation; genotype; human genetic material tag; inborn aminoacid metabolism disorder; mental retardation; molecular site; mutant; nuclear magnetic resonance spectroscopy; oxidoreductase; phenotype; single strand conformation polymorphism; site directed mutagenesis; spectrometry; thermodynamics

The long term aims of this research program have been to relate the basis biochemistry of electron transfer flavoprotein (ETF) and electron transfer flavoprotein-ubiquinone oxidoreductase (ETF)-CO) to a human inborn error of metabolism, glutaric acidemia type II (GA2), and to obtain a better understanding of the normal proteins from investigations of defects in these proteins. GA2 results in defective oxidation of fatty acids and amino acids and is often fatal. The objective of the proposed research is to establish the molecular bases, aat the nucleotide and protein levels, of ETF deficiency. We will (a) identify mutations in patients with GA2 due to ETF deficiency; (b) express selected mutations i E> coli; (c) investigate the kinetic, redox and structural properties of the mutant ETFs and (d) solve the crystal structure of human ETF. We developed two systems to express ETFs in E. coli. The first expresses Paracoccus denitrificans ETF. Subunits of this bacterial ETF have 72% overall sequence similarity (about 60% sequence identity) with the human subunits. This system will be used to investigate the covalent binding of an FAD analog, 8-chloroFAD to the apoETF. The second, recently developed system expresses human ETF using the expression vector originally developed for the bacterial protein, and will be used to express human mutations. Other site-directed mutations based on modification of human ETF with photoaffinity and other FAD analogs that bind covalently tot the apoprotein will extend these investigations of the basic biochemistry of ETF beyond those indicated by naturally occurring mutations. These investigations aim to define the FAD binding site and docking sites for the flavoprotein dehydrogenases and electron transfer flavoprotein oxidoreductase. The kinetic and redox behavior of mutant ETFs will be investigated to determine whether mutations affect the rate of electron transfer through the protein or whether they affect the flavin redox potential, so that electron transfer through the protein becomes thermodynamically unfavorable. Structural studies on normal and mutant ETFs will include 13C, 15N and 31P NMR, circular dichroism and fluorescence spectroscopic investigations and will be complemented by crystallographic investigations which are also in progress.

该研究计划的长期目的是联系基础 电子转移黄蛋蛋白（ETF）和电子转移的生物化学 黄蛋白 - 泛酮氧化还原酶（ETF）-CO） 代谢，II型谷氨酸血症（GA2），并获得更好的 从研究中了解正常蛋白的缺陷 这些蛋白质。 GA2导致脂肪酸和 氨基酸，通常是致命的。 拟议的研究的目的是 为了建立分子碱基，核苷酸和蛋白质水平， ETF缺乏。 我们将（a）确定由于GA2患者的突变 ETF缺乏症； （b）明确选定的突变i e e> coli; （c）调查 突变ETF和（d）的动力学，氧化还原和结构特性 解决人类ETF的晶体结构。 我们开发了两个系统来 大肠杆菌中的Extfs ETF。 第一个表达denitrificans etf。 该细菌ETF的亚基具有72％的总序列相似性（关于 与人类亚基的60％序列身份）。 该系统将被使用 研究FAD类似物的共价结合，8-氯弗加 apoetf。 第二个，最近开发的系统使用 最初为细菌蛋白开发的表达载体， 将用于表达人类突变。 其他站点定向的突变 基于对人类ETF的修饰，具有光接触和其他FAD类似物 这种约束共价tot apoprotein将扩展这些研究 ETF的基本生物化学超出了自然发生的生物化学 突变。 这些调查旨在定义FAD结合位点，并 黄蛋蛋白脱氢酶和电子转移的对接位点 黄素蛋白氧化还原酶。 突变体的动力学和氧化还原行为 将研究ETF，以确定突变是否影响 电子通过蛋白质转移或是否影响黄素 氧化还原电位，使电子通过蛋白质转移变为 热力学上不利。 正常和突变体的结构研究 ETF将包括13C，15N和31p NMR，圆形二色性和荧光 光谱研究，将由晶体学补充 也正在进行的调查。