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: 8362151
• 负责人: DAVID B. GOODIN
• 金额: $ 0.25万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8362151
• 关键词: Active Sites; Affinity; Automobile Driving; Behavior; Binding; Camphor 5-Monooxygenase; Cytochromes; Data; Electron Transport; Electrons; Enzymes; Funding; Grant; Heme; Injection of therapeutic agent; Libraries; Molecular; Molecular Evolution; National Center for Research Resources; Oxidation-Reduction; Pathway interactions; Peptides; Peroxidases; Principal Investigator; Proteins; Pterins; Radiation; Research; Research Infrastructure; Resources; Series; Site; Source; Structure; Substrate Specificity; Sushi Domain; United States National Institutes of Health; Variant; Withdrawal; analog; base; cofactor; cost; design; human NOS2A protein; mutant; novel; novel strategies; oxidation; programs; structural biology; success

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 作为NIH资助的项目的一部分，我们正在开发使用合成的分子电线？，底物类似物的底物类似物束缚于光敏剂或亲和力标签，以研究几种血红素酶，包括细胞色素P450，过氧化物酶，以及可诱导的一氧化氮合酶（ iNos）。这些电线的设计目的是专门与目标酶的活性位点结合。一旦结合，这些结构可用于光化学触发从氧化还原活性血红素辅因子中注入或退出电子，以允许以新颖的方式触发机制和营业额。此外，这些束缚的底物类似物可用于从突变蛋白库中选择差异结合行为，从而使新的底物特异性分子演化采用了新的方法。对于该程序的成功及其关键特征之一是这些人造线与目标结合的结构表征。在初步研究中，我们已经成功地获得了与P450CAM活跃位点结合的一系列合成线变体的结构。这包括电线的底物，接头和敏化器部分的变化，这些结构在推动我们开发P450的新型底物氧化的努力方面已证明是无价的。我们还获得了与CCP的电子传递通道突变体结合的基于肽的分子线的初步结构，该结构证明我们将能够用合成结构代替酶中的电子传递途径。最后，我们最近获得了在基于合成翼蛋白的电线的存在下生长的iNOS晶体的数据，这些晶体显示了在酶的翼展部位结合的证据。我们建议通过确定与这三种酶的WT和突变体形式结合的较大电线的结构来扩展这些初始努力。