STRUCTURE/FUNCTION AND PROTEIN-PROTEIN INTERACTION ANALYSIS IN HG DETOXIFICATION

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 代表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。 在这个项目中，我们正在研究蛋白质-蛋白质相互作用在无机汞和有机汞的细菌解毒途径的关键蛋白质之间的金属离子转移机制中的作用，并阐明嵌入蛋白质结构中的化学和物理性质，这些结构有助于将Hg（II）直接转移到还原解毒的必需酶。已经确定了该途径的四个组分的结构，并且正在追求一个完整的膜转运蛋白的结构，以促进蛋白质的结构/功能分析。 连同结构信息，我们使用定点诱变来评估改变的结构特征对几种蛋白质的个别性质和功能及其相互作用的影响。 这些研究的目的是揭示从进化的角度来看对于这种解毒途径有效至关重要的蛋白质的特征。 质谱已被证明是非常宝贵的，在验证氧化和蛋白质水解问题与我们的几个突变的酶，并在验证金属离子络合物，我们已经在我们的结构和动力学研究中使用的形成。

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. In this project we are investigating the role of protein-protein interactions in the mechanism of metal ion transfers between the key proteins of the bacterial detoxification pathway for inorganic mercury and organomercurials and elucidating the chemical and physical properties embedded in the protein structures that facilitate the directed transfer of Hg(II) to the essential enzyme for reductive detoxification. The structures of four components of the pathway have been determined and the structure of an integral membrane transport protein is being pursued to facilitate structure/function analysis of the proteins. Together with the structural information we use site-directed mutagenesis to evaluate the effects of altered structural features on the individual properties and functions of the several proteins and on their interactions. The goal of the studies is to reveal the features of the proteins that are essential from an evolutionary perspective for this detoxification pathway to be effective. Mass spec has proved invaluable in verifying oxidation and proteolysis problems with several of our mutated enzymes and in verifying formation of metal ion complexes that we have used in our structural and kinetic studies.