Structure Determination of Membrane Proteins in Phospholipid Bilyaers

描述(由申请者提供)：有三个主要目标。一是继续发展一种在生理条件下测定磷脂双层膜蛋白结构的通用方法。这一目标很重要，因为膜蛋白是结构确定的高优先级目标，而现有方法对这类蛋白有很大的局限性。第二个目标是将结构测定方法应用于细菌汞解毒系统的汞转运膜蛋白。首先将确定MerF和meel的结构，每个都有两个跨膜(TM)螺旋，然后将继续研究这个家族中具有三个(Mert)和四个(Merc)TM螺旋的其他成员。研究这一蛋白质家族在研究中起到了第二个作用，为固体核磁共振波谱仪器和实验方法的发展提供了越来越大和复杂的蛋白质靶标。这些蛋白质之间的比较可能会提供一些见解，为什么能够解毒汞(II)的独立菌株有不同数量的运输蛋白，这些蛋白质有不同数量的TM螺旋，以及这些蛋白质有不同数量的半胱氨酸残基与汞结合。第三个目标来自技术的发展和对这一蛋白质家族成员的结构发现，这为汞转运膜蛋白与我们先前确定的周质蛋白MERP和汞还原酶N端的“MERP样”结构域(其结构已由他人确定)的二元和三元复合体的组装和结构研究奠定了基础。汞还原酶(MERA)将剧毒的汞(II)还原为毒性较小、挥发性较低的汞(0)，并被动地扩散到细胞外。将汞(II)从周质运输到细胞质是一个关键步骤，必须严格控制，使高活性的汞(II)在溶液中永远不是游离的，并可与基本细胞蛋白上的半胱氨酸残基反应，这是其在没有聚合体操纵子的细胞中毒性的来源。我们的研究方法是跨学科和综合性的，包括分子生物学、生物化学、样品制备、核磁共振仪器的构建和改进、核磁共振实验的开发和执行以及结构计算。汞转运膜蛋白本身及其功能复合体的结构为汞(II)跨双层膜转运机制的功能研究奠定了基础。这些研究的结果有可能影响人类急性汞中毒的治疗，这是将结构生物学方法应用于环境研究的首批例子之一，因为器官汞化合物在食物供应(特别是大型鱼类)和环境中广泛分布。 与公共卫生相关：通过开发一种在天然磷脂双层环境中确定膜蛋白结构的通用方法，我们将能够解决生物医学研究中的重要问题。这项技术的发展将产生广泛的影响，因为大多数治疗药物都是针对驻留在细胞膜上的蛋白质受体。本提案中描述的研究主要集中在细菌汞解毒系统的汞转运膜蛋白上。它们的结构可能有助于发现人类汞中毒的解毒剂，从更广泛的角度来看，它们提供了将结构生物学应用于环境问题的机会。

DESCRIPTION (provided by applicant): There are three principal goals. The first is to continue the development of a general method for determining the structures of membrane proteins in phospholipid bilayers under physiological conditions. This goal is important because membrane proteins are high priority targets for structure determination, and existing methods have substantial limitations for this class of proteins. The second goal is to apply the method for structure determination to the mercury transport membrane proteins of the bacterial mercury detoxification system. The structures of MerF and MerE, each of which have two trans membrane (TM) helices, will be determined first, and then the research will proceed to other members of this family with three (MerT) and four (MerC) TM helices. Studying this family of proteins serves a second role in the research by providing protein targets of increasing size and complexity as challenges for the development of the instrumentation and experimental methods of solid-state NMR spectroscopy. Comparisons among these proteins may provide insights into why independent isolates of bacteria capable of detoxifying Hg(II) have varying numbers of transport proteins, the proteins have different numbers of TM helices, and the proteins have different numbers of pairs of cysteine residues that bind mercury. The third goal follows from the development of the technology and the structural findings on members of this family of proteins, which sets the stage for the assembly and structural studies of binary and ternary complexes of examples of the mercury transport membrane proteins with the periplasmic protein, MerP, whose structure we determined previously, and the N-terminal "MerP-like" domain of mercuric reductase, MerA, whose structure has been determined by others. Mercuric reductase (MerA) reduces the highly toxic Hg(II) to the less toxic and volatile Hg(0) that passively diffuses out of the cells. Transporting the Hg(II) from the periplasm to the cytoplasm is a key step and it must be tightly controlled so that the highly reactive Hg(II) is never free in solution and available for reaction with the cysteine residues on essential cellular proteins, which is the source of its toxicity in cells without the mer operon. Our research approach is interdisciplinary and comprehensive, encompassing molecular biology, biochemistry, sample preparation, construction and modification of NMR instrumentation, the development and execution of NMR experiments, and structure calculations. The structures of the mercury transport membrane proteins alone and in their functional complexes set the stage for functional studies of the mechanism of transporting Hg(II) across the bilayer membrane. The results of these studies have the potential to impact the treatment of acute mercury toxicity in humans, and this is one of the first examples of applying the methods of structural biology to environmental research because of the widespread distribution of organ mercurial compounds in the food supply (especially in large fish) and the environment. PUBLIC HEALTH RELEVANCE: By developing a general method for determining the structures of membrane proteins in their native phospholipid bilayer environment we will be able to tackle important problems in biomedical research. The development of this technology will have a broad impact since the majority of therapeutic drugs are targeted to protein receptors that reside in cell membranes. The studies described in this proposal are focused on the mercury transport membrane proteins of the bacterial mercury detoxification system. Their structures may assist in the discovery of antidotes to mercury poisoning in humans, and from a broader perspective they provide an opportunity to apply structural biology to an environmental problem.