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的结构上，以及它在激活超氧化物生成系统时如何改变。 该提案中提出的基本假设是，该细胞色素是超氧化物产生的中央电子转移酶，其结构的改变将调节电子跨质膜的流动。 阐明该电子流量调节的基本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。研究该蛋白质中现有的结构/功能关系可能会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微生物杀伤。 更具体地说，该提案概述了使新型单克隆和重组抗体识别天然黄素色素B的表位的策略，从而定义了其在1-5DELTA范围内的三维结构，并将其放置在10 -60 Delta范围内。 它描述了一个共价修改黄素色素B的计划，以便用荧光探针标记其表面上的不同位点，识别可能的磷酸化位点，并识别血红素结合位点的位置。 为了准确识别这些修饰，它建议将HPLC与修饰蛋白的蛋白水解消化物的MALDI-TOF质谱和肽测序结合使用。 使用这些新的探针和使用荧光探针对黄素色素的共价修饰，可以使用荧光谐振能传递相对于其一级结构及其内在的HEMES绘制蛋白质表面。该提案还概述了一种使用多维NMR技术的策略，包括转移的Noesy，Tr Roesy，Tocsy，Rosey方法来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，以揭示可以在噬菌体上显示的黄素色素B结构元素，以筛选以筛选结合伴侣或单核抗体。 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序列，跨膜拓扑和高分辨率的离散表面位点的三维结构。