NEUTROPHIL CYTOCHROME B STRUCTURE/FUNCTION

中性粒细胞色素 B 结构/功能

• 批准号: 6169782
• 负责人: ALGIRDAS JOSEPH JESAITIS
• 金额: $ 22.96万
• 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 2004-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6169782
• 关键词: cell membrane; conformation; cytochrome b; electron transport; fluorescence resonance energy transfer; fluorescent dye /probe; genetic library; heme; hemoprotein; high performance liquid chromatography; human tissue; laboratory rabbit; mass spectrometry; membrane proteins; messenger RNA; monoclonal antibody; neutrophil; nuclear magnetic resonance spectroscopy; oxidation; phosphorylation; physical model; protein sequence; protein structure function; superoxides

The broad long term objective of this proposal is to determine the molecular basis of superoxide production in human neutrophils. The proposed investigation will focus on the structure of human neutrophil flavocytochrome b and how it changes upon activation of the superoxide generating system. The fundamental assumption made in this proposal is that this cytochrome is the central electron transferase of superoxide production and that alterations of its structure will regulate the flow of electrons across the plasma membrane. Elucidation of the fundamental 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 protein 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 making novel monoclonal and recombinant antibodies recognizing native flavocytochrome b epitopes, defining their three dimensional structure in the 1-5delta range, and their placement in the protein in the 10 -60 delta range. It describes a plan to covalently modify flavocytochrome b in order to mark different sites on its surface with fluorescent probes, identify possible phosphorylation sites, and identify the locations of the heme binding sites. To precisely identify these modifications, it proposes to use HPLC combined with MALDI-TOF mass spectrometry and peptide sequencing of proteolytic digests of the modified proteins. Using these new probes and covalent modification of the flavocytochrome with fluorescent probes, it will be possible to map the protein surface relative to its primary structure and its intrinsic hemes using fluorescence resonance energy transfer. The proposal also outlines a strategy to use multidimensional NMR techniques including Transferred NOESY, Tr ROESY, TOCSY, ROSEY methods to determine the antibody-bound structures of peptide mimetics of the cytochrome surface. Finally a gene fragment phage expression library of the gp91 phox and p22phox genes will be produced to reveal flavocytochrome b structural elements that can be displayed on phage for screening against binding partners or monoclonal antibodies. Successful completion of this work will permit the development of a structural model of the cytochrome that will be able to incorporate its known sequence, transmembrane topology, and high resolution three dimensional structure of discrete surface sites.

该提案的广泛长期目标是确定人类中性粒细胞中超氧化物产生的分子基础。 拟议的研究将集中于人中性粒细胞黄素细胞色素 b 的结构以及它在超氧化物生成系统激活后如何变化。 该提议的基本假设是，这种细胞色素是超氧化物产生的中心电子转移酶，其结构的改变将调节电子穿过质膜的流动。 阐明这种电子流调节的基本结构机制将为理解吞噬细胞介导的杀微生物和组织损伤的基本过程的分子基础提供必要的关键信息。对该蛋白质中存在的结构/功能关系的检查可能会导致合理设计的药物的开发，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灭微生物。 更具体地说，该提案概述了制备识别天然黄细胞色素 b 表位的新型单克隆和重组抗体的策略，定义了它们在 1-5delta 范围内的三维结构，以及它们在 10 -60 delta 范围内的蛋白质中的位置。 它描述了共价修饰黄素细胞色素 b 的计划，以便用荧光探针标记其表面的不同位点，识别可能的磷酸化位点，并识别血红素结合位点的位置。 为了精确识别这些修饰，建议使用 HPLC 结合 MALDI-TOF 质谱和肽测序对修饰蛋白的蛋白水解消化物进行分析。 使用这些新探针以及用荧光探针对黄素细胞色素进行共价修饰，将有可能利用荧光共振能量转移来绘制蛋白质表面相对于其一级结构及其内在血红素的图谱。该提案还概述了使用多维核磁共振技术（包括转移的 NOESY、Tr ROESY、TOCSY、ROSEY 方法）的策略，以确定细胞色素表面的肽模拟物的抗体结合结构。 最后，将产生 gp91 phox 和 p22phox 基因的基因片段噬菌体表达文库，以揭示黄素细胞色素 b 结构元件，这些元件可以展示在噬菌体上，用于筛选结合伴侣或单克隆抗体。 这项工作的成功完成将允许开发细胞色素的结构模型，该模型将能够结合其已知的序列、跨膜拓扑和离散表面位点的高分辨率三维结构。