NEUTROPHIL FLAVOCYTOCHROME B STRUCTURE AND FUNCTION

中性粒细胞黄细胞色素 B 的结构和功能

• 批准号: 6928320
• 负责人: ALGIRDAS JOSEPH JESAITIS
• 金额: $ 34.34万
• 依托单位: MONTANA STATE UNIVERSITY - BOZEMAN
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-03-01 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6928320
• 关键词: NAD(P)H dehydrogenase; clinical research; cryoelectron microscopy; cytochrome b; electron transport; enzyme induction /repression; epitope mapping; flavonoids; fluorescence resonance energy transfer; fluorescent dye /probe; high performance liquid chromatography; human subject; human tissue; immunologic assay /test; laboratory mouse; laboratory rabbit; leukocyte oxidative burst; liquid chromatography mass spectrometry; membrane proteins; membrane structure; monoclonal antibody; neutrophil; phage display; protein purification; protein structure function; superoxides

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.

描述（由申请人提供）：该提案的广泛长期目标是了解人类嗜中性粒细胞中超氧化物产生的分子基础。拟议的研究将重点放在人类嗜中性粒细胞色素B（CYTB）激活后的结构变化上。该提案中提出的基本假设是，该电子转移酶结构的改变会调节电子跨膜跨膜的流动。阐明该电子流量调节的结构机制将提供至关重要的信息，以了解吞噬细胞介导的微生物杀伤和组织损伤中基本过程的分子基础。对这种整体膜糖蛋白中现有的结构/功能关系的检查可能导致开发合理设计的药物，这些药物可以改善中性粒细胞介导的组织损伤并增强中性粒细胞介导的杀伤性杀伤。更具体地说，该提案概述了利用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素色素B的表位，从而定义了它们放置在黄酮色素b的表面上。它描述了一个计划，可以使用荧光共振能传递，测量抗体位点之间的距离，并确定这些距离在激活氧化酶时如何变化，以共价修改这些抗体，以使用荧光探针进行三角剖分血红素位点。该提案还概述了确定表面拓扑和分子内接近的策略。该策略包括纯化的黄瓜色素的选择性交联，遵循有限的蛋白水解和HPLC/质谱分析。它还概述了通过单分子常规和冷冻电子显微镜分析CYTB分子形状的计划。最后，该提案利用了抗体印记中取得的最新进展，这是主要研究者开发的噬菌体显示分析的应用，该分析确定了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的邻居图，以帮助设计抗体 - 抗原相互作用的肽抑制剂，并生产原料，以使抗体肽复合物在其他支持下制造抗体肽复合物，以确定共晶和结构。这项工作的成功完成将启动黄素色素的结构模型的发展，该模型将能够结合其已知的序列，跨膜拓扑，总分子形状和离散表面位点的抗体烙印结构，对于任何完全解决的X射线晶体结构的解释至关重要。