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

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 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细菌解毒途径的关键蛋白质和有机汞之间的金属离子转移机制中的作用，并阐明蛋白质结构中嵌入的化学和物理性质，促进汞(II)直接转移到还原解毒的关键酶。已经确定了该途径的四个组成部分的结构，并正在寻找一个完整的膜运输蛋白的结构，以便于对这些蛋白的结构/功能进行分析。结合结构信息，我们正在使用定点突变来评估结构特征改变对几种蛋白质的个别性质和功能以及它们之间相互作用的影响。这项研究的目的是从进化的角度揭示这种解毒途径有效所必需的蛋白质的特征。事实证明，质谱仪在验证我们的几种突变酶的氧化和蛋白质分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具有极高的价值。

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 spectrometry 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.