CHARACTERIZATION OF MOLECULAR WIRES BOUND TO P450CAM, CCP, AND INOS

与 P450CAM、CCP 和 INOS 结合的分子线的表征

• 批准号: 8170093
• 负责人: DAVID B. GOODIN
• 金额: $ 0.51万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8170093
• 关键词: Active Sites; Affinity; Automobile Driving; Behavior; Binding; Camphor 5-Monooxygenase; Computer Retrieval of Information on Scientific Projects Database; Cytochromes; Data; Electron Transport; Electrons; Enzymes; Funding; Grant; Heme; Injection of therapeutic agent; Institution; Libraries; Molecular; Molecular Evolution; Oxidation-Reduction; Pathway interactions; Peptides; Peroxidases; Proteins; Pterins; Research; Research Personnel; Resources; Series; Site; Source; Structure; Substrate Specificity; United States National Institutes of Health; Variant; Withdrawal; analog; base; cofactor; design; human NOS2A protein; mutant; novel; novel strategies; oxidation; programs; success

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. As part of an NIH funded project, we are developing the use of synthetic ?molecular wires?, substrate analogs tethered to photo-sensitizers or affinity tags, for the study of several heme enzymes including cytochrome P450s, peroxidases, and inducible nitric oxide synthase (iNOS). These wires are being designed to bind specifically to the active site of the target enzyme. Once bound, these structures may be used to photochemically trigger the injection or withdrawal of electrons from the redox active heme cofactor to allow the mechanism and turnover to be triggered in a novel way. In addition, these tethered substrate analogs may be used to select for differential binding behavior from a library of mutant proteins, allowing a new approach to the molecular evolution of novel substrate specificity. Crucial to the success of this program, and one of its key features, is the structural characterization of these artificial wires bound to their targets. In preliminary studies, we have been successful in obtaining structures of a series of synthetic wire variants bound to the active site of P450cam. This includes variations in the substrate, linker and sensitizer portion of the wire, and these structures have proven invaluable in driving our efforts to evolve P450s for novel substrate oxidation. We have also obtained a preliminary structure of a peptide based molecular wire bound to an electron transfer channel mutant of CcP, which demonstrates that we will be able to replace the electron transfer pathway in the enzyme with synthetic structures. Finally, we have recently obtained data on iNOS crystals grown in the presence of synthetic pterin based wires that show evidence of binding at the pterin site of the enzyme. We propose to extend these initial efforts by determining structures of a larger array of wires bound to WT and mutant forms of these three enzymes.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 作为 NIH 资助项目的一部分，我们正在开发使用合成“分子线”、与光敏剂或亲和标签相连的底物类似物，用于研究几种血红素酶，包括细胞色素 P450、过氧化物酶和诱导型一氧化氮合酶（诱导型一氧化氮合酶）。这些导线被设计为特异性结合到目标酶的活性位点。一旦结合，这些结构可用于通过光化学方式触发氧化还原活性血红素辅因子的电子注入或撤回，从而以新颖的方式触发该机制和周转。此外，这些束缚底物类似物可用于从突变蛋白库中选择差异结合行为，从而为新底物特异性的分子进化提供新方法。该计划成功的关键及其关键特征之一是这些与目标结合的人造导线的结构特征。在初步研究中，我们已经成功获得了一系列与 P450cam 活性位点结合的合成线变体的结构。这包括导线的底物、连接体和敏化剂部分的变化，并且这些结构已被证明对于推动我们开发 P450 以实现新型底物氧化的努力具有无价的价值。我们还获得了与 CcP 电子传递通道突变体结合的基于肽的分子线的初步结构，这表明我们将能够用合成结构取代酶中的电子传递途径。最后，我们最近获得了在合成蝶呤基线存在下生长的 iNOS 晶体的数据，这些数据显示了在酶的蝶呤位点上结合的证据。我们建议通过确定与 WT 结合的更大阵列的线的结构以及这三种酶的突变形式来扩展这些初步努力。