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

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 表示子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 作为美国国立卫生研究院资助项目的一部分，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（iNOS）。这些金属丝被设计成与靶酶的活性位点特异性结合。一旦结合，这些结构可以用于光化学触发电子从氧化还原活性血红素辅因子的注入或撤回，以允许以新的方式触发机制和周转。此外，这些拴系的底物类似物可用于从突变蛋白质文库中选择差异结合行为，从而为新型底物特异性的分子进化提供了新的方法。该计划成功的关键，也是其关键特征之一，是这些与目标结合的人造线的结构特征。在初步的研究中，我们已经成功地获得了一系列的合成线变体结合到P450 cam的活性位点的结构。这包括衬底，连接器和敏化剂部分的线的变化，这些结构已被证明是无价的，在推动我们的努力，以发展P450的新的衬底氧化。我们还获得了一个初步的结构的肽为基础的分子线绑定到电子转移通道突变体的CcP，这表明，我们将能够取代电子转移途径的酶与合成结构。最后，我们最近获得的数据iNOS晶体生长在合成的蝶呤为基础的电线，显示在蝶呤网站的酶结合的证据。我们建议通过确定与WT和这三种酶的突变形式结合的更大阵列的电线的结构来扩展这些初步的努力。

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.