NEUTROPHIL FLAVOCYTOCHROME B STRUCTURE AND FUNCTION

描述(由申请人提供)：这项提案的广泛长期目标是了解人类中性粒细胞产生超氧化物的分子基础。拟议的研究将集中在人中性粒细胞黄色素b(Cytb)在NADPH氧化酶激活时的结构变化。这一提议的基本假设是，这种电子转移酶结构的变化调节着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为理解吞噬细胞介导的杀灭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。对这种完整的膜糖蛋白结构/功能关系的研究可能有助于开发合理设计的药物，以减轻中性粒细胞介导的组织损伤，增强中性粒细胞介导的杀菌作用。更具体地说，这项建议概述了利用9种能够识别独特的天然黄色素b表位的单抗和重组抗体的策略，定义了它们在黄色素b表面的位置。它描述了一项计划，用荧光探针共价修饰这些抗体，使用荧光共振能量转移来三角测量血红素位置，测量抗体位置之间的距离，并确定这些距离在酶激活时如何变化。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。这一策略包括对纯化的黄色素进行选择性交联化，然后进行有限蛋白分解和高效液相色谱/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，该建议利用了抗体印迹技术的最新进展，这是首席研究员开发的噬菌体展示分析的一种应用，它识别抗体结合部位的结构元素。这些信息将被用来帮助制作抗原表位的最近邻图，以帮助设计抗体-抗原相互作用的多肽抑制剂，并为制备抗体-多肽复合物提供原料，用于在其他载体下进行共结晶和结构测定。这项工作的成功完成将启动黄色素结构模型的开发，该模型将能够包含其已知的序列、跨膜拓扑、总分子形状和离散表面位置的抗体印迹结构，并对解释任何完全溶解的X射线晶体结构至关重要。

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.