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

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 作为NIH资助的一个项目的一部分，我们正在开发使用合成分子导线，即与光敏剂或亲和标签捆绑在一起的底物类似物，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)。这些线被设计成专门与靶标酶的活性部位结合。一旦结合，这些结构可以用来光化学地触发电子从氧化还原活性血红素辅因子中的注入或退出，从而允许以一种新的方式触发机制和翻转。此外，这些拴系底物类似物可用于从突变蛋白质库中选择不同的结合行为，从而允许一种新的方法来研究新底物特异性的分子进化。这一计划成功的关键，也是其关键特征之一，是这些捆绑在目标上的人造电线的结构特征。在初步研究中，我们已经成功地获得了一系列与P450cam活性部位结合的合成线变体的结构。这包括金属丝的衬底、连接物和感光剂部分的变化，这些结构已被证明在推动我们为新型衬底氧化进化P450方面的努力方面具有无价的价值。我们还获得了与CCP的电子转移通道突变体结合的多肽分子线的初步结构，这表明我们将能够用合成结构取代酶中的电子转移途径。最后，我们最近获得了在合成的蝶呤基导线中生长的iNOS晶体的数据，这些数据表明有证据表明iNOS晶体与酶的蝶呤蛋白结合。我们建议通过确定与WT结合的更大数量的导线的结构和这三种酶的突变形式来扩展这些最初的努力。

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.