NEUTROPHIL FLAVOCYTOCHROME B STRUCTURE AND FUNCTION

描述（由申请人提供）：本提案的长期目标是了解人类中性粒细胞超氧化物产生的分子基础。拟建的研究将集中于人中性粒细胞黄细胞色素b （Cytb）在NADPH氧化酶激活后的结构变化。这个提议的基本假设是，这种电子转移酶结构的改变调节了电子在它所跨越的膜上的流动。阐明这种电子流调控的结构机制，将为理解吞噬细胞介导的杀微生物和组织损伤这一重要过程的分子基础提供必要的信息。研究这种整体膜糖蛋白的结构/功能关系可能有助于开发合理设计的药物，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作用。更具体地说，本提案概述了开发9种单克隆和重组抗体的策略，这些抗体识别独特的天然黄细胞色素b表位，定义它们在黄细胞色素b表面的位置。它描述了用荧光探针共价修饰这些抗体的计划，使用荧光共振能量转移对血红素位点进行三角测量，测量抗体位点之间的距离，并确定这些距离在氧化酶激活时如何变化。该提案还概述了一种确定表面拓扑和分子内接近度的策略。该策略包括选择性交联纯化黄细胞色素，然后进行有限的蛋白水解和HPLC/质谱分析。它还概述了用单分子常规显微镜和低温电子显微镜分析Cytb分子形状的计划。最后，该提案利用了抗体印迹的最新进展，这是一种由首席研究员开发的噬菌体展示分析的应用，可以识别抗体结合位点的结构元件。该信息将用于帮助生成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，并生产用于在其他支持下共结晶和结构确定的抗体-肽复合物的原料。这项工作的成功完成将启动黄细胞色素结构模型的发展，该模型将能够结合其已知的序列、跨膜拓扑结构、总分子形状和离散表面位点的抗体印记结构，并且对任何完全解决的x射线晶体结构的解释至关重要。

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.