STUDIES ON ELECTRON TRANSFER DOMAINS AND COMPLEXES OF FLAVOPROTEINS

这项研究计划的长期目标一直是将基础 电子转移黄素蛋白(ETF)的生物化学及电子转移 黄素蛋白-泛醌氧化还原酶(ETF)-CO)导致人类先天错误 代谢，戊二酸血症II型(GA2)，并获得较好的 从缺陷研究中对正常蛋白质的认识 这些蛋白质。GA2会导致脂肪酸的氧化缺陷和 氨基酸，往往是致命的。拟议研究的目标是 为了在核苷酸和蛋白质水平上建立分子基础， ETF缺乏症。我们将(A)确定GA2患者由于以下原因而发生突变 ETF缺乏症；(B)在E&GT；Coli中表达选定的突变；(C)调查 突变ETF和(D)的动力学、氧化还原和结构性质 解决人的ETF的晶体结构。我们开发了两个系统来 在大肠杆菌中表达ETF。第一个表达的是脱氮副球藻ETF。 该细菌ETF的亚基具有72%的总序列相似性(约 60%的序列同源性)。这个系统将被使用 为了研究FAD类似物8-氯FAD与 ApoETF。第二个是最近开发的系统，它使用 该表达载体最初是为细菌蛋白开发的，以及 将被用来表达人类突变。其他定点突变 基于用光亲和力和其他时尚类似物修饰人ETF 与脱辅基蛋白共价结合将扩展这些研究 ETF的基础生物化学超出了自然发生的范围 突变。这些研究旨在确定FAD结合位点和 黄素蛋白脱氢酶与电子转移的对接位置 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，以确定突变是否影响 通过蛋白质的电子转移或它们是否影响黄素 氧化还原电位，因此通过蛋白质的电子转移成为 热力学上是不利的。正常和突变的结构研究 ETF包括~(13)C、~(15)N和~(31)P核磁共振、圆二色谱和荧光 光谱研究，并将得到结晶学的补充 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.