ELECTRON TRANSFER FLAVOPROTEIN AND GLUTARIC ACIDEMIA II

这项研究计划的长期目标是将基本的 电子传递黄素蛋白生物化学与电子传递 黄素蛋白-泛醌氧化还原酶（ETF-Q 0）与人类先天性缺陷 代谢，谷氨酸血症II型（GA 2），并获得更好的 通过对缺陷的研究来了解正常蛋白质， 这些蛋白质。 GA 2导致脂肪酸氧化缺陷， 氨基酸，往往是致命的。 拟议研究的目的是 在核苷酸和蛋白质水平上， ETF亏损 我们将（a）确定GA 2患者的突变， （B）在E.大肠杆菌;（c） 研究突变体的动力学、氧化还原和结构特性 （d）解析人ETF的晶体结构。 我们开发了两 在E.杆菌第一种是副球菌 黄金ETF 这种细菌ETF的子单位有72%的整体 与人的序列相似性（约60%序列同一性） 亚单位。 该系统将主要用于光谱 我们已经承诺的调查。 第二，最近 所开发的系统使用表达载体表达人ETF 最初是为细菌蛋白质开发的，并将用于 表达人类突变。 其他定点突变基于 用光亲和性和其它FAD类似物修饰人ETF， 与脱辅基蛋白共价结合将扩展这些研究， ETF的基本生物化学超出了自然发生的生物化学 突变。 这些研究旨在确定FAD结合位点， 黄素蛋白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 将对ETF进行调查，以确定突变是否会影响 电子通过蛋白质的转移，或者它们是否会影响黄素 氧化还原电位，因此通过蛋白质的电子转移变得 对你不利。 正常和突变体的结构研究 ETF将包括13 C、15 N和31 P NMR、圆二色谱和 荧光光谱研究，并将补充 晶体学研究也在进行中。

The long term aims of this research program have been to relate the basic biochemistry of electron transfer flavoprotein (ETF) and electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-Q0) 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, at 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 in 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 (approximately 60% sequence identity) with the human subunits. This system will be used primarily for spectroscopic investigations to which we have already committed. 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 to 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 of 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.