STUDIES ON ELECTRON TRANSFER DOMAINS AND COMPLEXES OF FLAVOPROTEINS

这项研究计划的长期目标是将基础 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）-CO）与人类先天性缺陷 代谢，谷氨酸血症II型（GA2），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。 GA2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）鉴定GA2患者的突变， ETF缺陷;（B）在大肠杆菌中表达选定的突变;（c）研究 突变ETF的动力学、氧化还原和结构特性，以及（d） 解析人类ETF的晶体结构 我们开发了两个系统， 在E.杆菌 第一种表达的是Paracoccus planficans ETF。 这种细菌ETF的亚基具有72%的总体序列相似性（约 60%序列同一性）。 该系统将用于 为了研究FAD类似物8-chlorofad与 apoETF。 第二，最近开发的系统使用 最初为细菌蛋白质开发的表达载体，和 将被用来表达人类的突变。 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 动力学和氧化还原 突变体行为 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、圆二色性和荧光 光谱研究，并将补充晶体学 调查也在进行中。

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.