NEUTROPHIL FLAVOCYTOCHROME B STRUCTURE AND FUNCTION

描述（由申请人提供）：本提案的广泛长期目标是了解人类中性粒细胞中超氧化物产生的分子基础。本研究主要关注NADPH氧化酶激活后人中性粒细胞黄细胞色素B（CytB）的结构变化。该提议的基本假设是，这种电子转移酶结构的改变调节了电子穿过它所跨越的膜的流动。阐明这种电子流调节的结构机制将提供必要的关键信息，以了解吞噬细胞介导的杀微生物剂杀伤和组织损伤的一个重要过程的分子基础。在这个完整的膜糖蛋白存在的结构/功能关系的检查可能会导致合理设计的药物，可以改善中性粒细胞介导的组织损伤和增强中性粒细胞介导的杀微生物剂的发展。更具体地说，该提案概述了利用9个单克隆和重组抗体，识别独特的天然黄细胞色素B表位，定义其在黄细胞色素B表面上的位置的策略。它描述了一个计划，以共价修饰这些抗体与荧光探针，三角血红素网站使用荧光共振能量转移，测量抗体位点之间的距离，并确定这些距离如何改变后激活的氧化酶。该提案还概述了一个战略，以确定表面拓扑结构和分子内的接近。该策略包括纯化的黄细胞色素的选择性交联，然后进行有限的蛋白水解和HPLC/质谱分析。它还概述了一个计划，分析Cytb分子的形状单分子常规和冷冻电子显微镜。最后，该提案利用了最近在抗体印迹方面取得的进展，这是由主要研究者开发的噬菌体展示分析的应用程序，可识别抗体结合位点的结构元素。该信息将用于帮助产生抗原表位的最近邻图，以帮助设计抗体-抗原相互作用的肽抑制剂，并产生用于制备抗体-肽复合物的原材料，用于在其他支持物下的共结晶和结构测定。这项工作的成功完成将启动开发的结构模型的flavocytochrome，将能够将其已知的序列，跨膜拓扑结构，总分子形状，和抗体印迹结构的离散表面站点和任何完全解决的X射线晶体结构的解释是必不可少的。

DESCRIPTION (provided by applicant): The broad long term objective of this proposal is to understand the molecular basis of superoxide production in human neutrophils. The proposed investigation will focus on the structural changes of human neutrophil flavocytochrome b (Cytb) upon activation of the NADPH oxidase. The fundamental assumption made in this proposal is that alterations in the structure of this electron transferase regulate the flow of electrons across the membrane it spans. Elucidation of the structural mechanism of regulation of this electron flow will provide crucial information necessary to understand the molecular basis of an essential process in phagocyte-mediated microbicidal killing and tissue injury. Examination of the structure/function relationships existing in this integral membrane glycoprotein may lead to the development of rationally designed drugs that could ameliorate neutrophil mediated tissue damage and enhance neutrophil mediated microbicidal killing. More specifically, this proposal outlines strategies for exploiting 9 monoclonal and recombinant antibodies that recognize unique native flavocytochrome b epitopes, defining their placement on the surface of flavocytochrome b. It describes a plan to covalently modify these antibodies with fluorescent probes, to triangulate heme sites using fluorescence resonance energy transfer, measure the distance between antibody sites, and determine how these distances change upon activation of the oxidase. The proposal also outlines a strategy to determine surface topology and intramolecular proximities. This strategy includes selective crosslinking of purified flavocytochrome followed limited proteolysis and HPLC/mass spectrometry analysis. It also outlines a plan to analyze Cytb molecular shape by single molecule conventional and cryoelectron microscopy. Lastly, the proposal exploits recent progress made in antibody imprinting, an application of phage display analysis developed by the Principal Investigator, which identifies structural elements of antibody binding sites. This information will be used to help produce a nearest neighbor map of the antigen epitope to aid in design of peptide inhibitors of the antibody-antigen interactions and produce raw material for making antibody-peptide complexes for cocrystallization and structure determination under other support. Successful completion of this work will initiate the development of a structural model of the flavocytochrome that will be able to incorporate its known sequence, transmembrane topology, gross molecular shape, and antibody imprint structure of discrete surface sites and be essential to the interpretation of any fully solved x-ray crystal structure.