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

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。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细菌解毒途径的关键蛋白质之间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，这些结构促进了汞（II）直接转移到还原性解毒的必需酶。该途径的四个组成部分的结构已经被确定，一个完整的膜运输蛋白的结构正在被追求，以促进蛋白质的结构/功能分析。结合结构信息，我们正在使用位点定向诱变来评估结构特征改变对几种蛋白质的个体特性和功能及其相互作用的影响。这些研究的目的是揭示蛋白质的特征，从进化的角度来看，这些蛋白质对这种解毒途径是有效的。质谱在验证我们的几种突变酶的氧化和蛋白质水解问题以及验证我们在结构研究中使用的金属离子络合物的形成方面被证明是无价的。

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. 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 are using 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 studies.