NEUTROPHIL CYTOCHROME B STRUCTURE/FUNCTION

这项提议的长期目标是确定人类中性粒细胞超氧化物产生的分子基础。该研究将集中于人类中性粒细胞黄细胞色素b的结构及其在超氧化物生成系统激活后的变化。本研究提出的基本假设是，细胞色素是产生超氧化物的中心电子转移酶，其结构的改变将调节电子在质膜上的流动。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重要过程的分子基础提供必要的信息。检查该蛋白中存在的结构/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杀微生物作用。更具体地说，本提案概述了制造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1-5 δ范围内的三维结构，以及它们在10 -60 δ范围内的蛋白位置。它描述了一种共价修饰黄细胞色素b的计划，以便用荧光探针标记其表面的不同位点，确定可能的磷酸化位点，并确定血红素结合位点的位置。为了准确鉴定这些修饰，建议使用HPLC结合MALDI-TOF质谱法和修饰蛋白水解酶切的肽段测序。利用这些新的探针和用荧光探针对黄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初级结构和内在血红素的地图。该提案还概述了一种使用多维核磁共振技术的策略，包括transfernoesy， Tr ROESY， TOCSY， ROSEY方法来确定细胞色素表面肽模拟物的抗体结合结构。最后构建gp91 phox和p22phox基因片段噬菌体表达文库，揭示可在噬菌体上显示的黄细胞色素b结构元件，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、跨膜拓扑结构和离散表面位点的高分辨率三维结构。

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.