Metalloenzymes and metal homeostasis

 描述(申请人提供)：这项研究计划集中在生物无机化学的两个领域：P1B-ATPase的金属运输和颗粒甲烷单加氧酶(PMMO)的生物甲烷氧化。这两个项目的共同主题包括了解完整的膜金属蛋白的结构和功能，阐明这些蛋白中金属位置的原子细节，以及建立金属离子转运或金属离子催化的分子机制。这两个过程是根本不同的：运输需要一个高度特定的金属结合部位，具有动态变化的亲和力，而催化需要一个专门为化学转化量身定做的部位。在这两种情况下，长期目标都是了解蛋白质支架的更大背景是如何赋予这些功能特性的。P1B-ATPase是一种完整的膜蛋白，它利用ATP水解的能量来跨膜运输金属离子，在所有生物体的金属动态平衡中发挥着关键作用。特别是，P1B-ATPase与人类的金属代谢疾病和人类病原体的毒力有关。尽管它们具有普遍的重要性，但与P1B-ATPase结构和功能相关的基本问题仍然没有解决，包括金属离子专一性的分子基础和转运机制。这些问题将通过表征一系列运输不同金属底物的P1B-ATPase来解决。实验方法包括生化表征、金属结合研究、光谱学、体外活性分析、活体分析、光谱学和结晶学。颗粒甲烷单加氧酶(PMMO)是一种低聚的、完整的膜金属酶，在甲烷营养细菌中将甲烷转化为甲醇，这些细菌利用甲烷作为唯一的碳和能源来源。甲烷氧化菌是减轻全球变暖对人类健康有害影响的潜在手段。此外，甲烷氧化菌可以氧化其他底物，包括卤代烃，因此成为生物修复应用的目标。主要问题是pMMO的结构和功能，包括活性中心的原子细节，氧和甲烷活化的化学机制，不同蛋白质亚基的作用，以及底物专一性的分子基础。实验方法包括天然pMMO、重组变异体pMMO和重组可溶性pMMO结构域的生化、光谱、力学和结晶学表征。

 DESCRIPTION (provided by applicant): This research program focuses on two areas of bioinorganic chemistry: metal transport by P1B-ATPases and biological methane oxidation by particulate methane monooxygenase (pMMO). Common themes of these two projects include understanding the structure and function of integral membrane metalloproteins, elucidating the atomic details of metal sites within these proteins, and establishing molecular mechanisms of metal ion transport or of catalysis by metal ions. These two processes are fundamentally different: transport requires a highly specific metal binding site with dynamically changing affinities whereas catalysis demands a site specifically tailored for chemical transformations. In both cases, the long term goal is to understand how the larger context of the protein scaffold confers these functional properties. The P1B-ATPases, integral membrane proteins that use the energy of ATP hydrolysis to transport metal ions across membranes, play a key role in metal homeostasis in all organisms. In particular, P1B-ATPases are linked to human diseases of metal metabolism and to the virulence of human pathogens. Despite their universal importance, fundamental issues related to P1B-ATPase structure and function remain unresolved, including the molecular basis of metal ion specificity and the mechanism of transport. These questions will be addressed by characterizing a range of P1B-ATPases that transport different metal substrates. Experimental approaches include biochemical characterization, metal binding studies, spectroscopy, in vitro activity assays, in vivo analysis, spectroscopy, and crystallography. Particulate methane monooxygenase (pMMO) is an oligomeric, integral membrane metalloenzyme that converts methane to methanol in methanotrophic bacteria, organisms that utilize methane as their sole source of carbon and energy. Methanotrophs are a potential means to mitigate the deleterious effects of global warming on human health. In addition, methanotrophs can oxidize other substrates, including halogenated hydrocarbons, and have therefore been targeted for bioremediation applications. Major questions central to pMMO structure and function will be addressed, including the atomic details of the active site, the chemical mechanisms of oxygen and methane activation, the roles of the different protein subunits, and the molecular basis for substrate specificity. The experimental approach involves biochemical, spectroscopic, mechanistic, and crystallographic characterization of native pMMOs, recombinant variant pMMOs, and recombinant soluble pMMO domains.