Structure Determination of Membrane Proteins in Phospholipid Bilyaers

磷脂胆汁中膜蛋白的结构测定

• 批准号: 8450700
• 负责人: STANLEY J OPELLA
• 金额: $ 27.73万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8450700
• 关键词: Acute; Antidotes; Bacteria; Binding; Biochemistry; Biomedical Research; Carrier Proteins; Cell membrane; Cells; Complex; Cysteine; Cytoplasm; Cytoplasmic Protein; Development; Devices; Diffuse; Docking; Drug Metabolic Detoxication; Drug Targeting; Environment; Family; Family Study; Family member; Fishes; Food Supply; Goals; Human; Magic; Membrane; Membrane Proteins; Membrane Transport Proteins; Mercury; Mercury (II) reductase; Mercury Poisoning; Methods; Micelles; Modification; Molecular Biology; N-terminal; NMR Spectroscopy; Operon; Organ; Periplasmic Proteins; Pharmaceutical Preparations; Phospholipids; Physiological; Preparation; Process; Protein Family; Proteins; Reaction; Research; Research Project Grants; Role; Sampling; Side; Site; Solutions; Source; Staging; Structure; System; Therapeutic; Toxic effect; base; insight; instrumentation; method development; novel strategies; periplasm; proteoliposomes; receptor; research study; solid state nuclear magnetic resonance; structural biology; technology development

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.

描述（由申请人提供）：有三个主要目标。首先是继续发展一种测定生理条件下磷脂双分子层膜蛋白结构的通用方法。这一目标很重要，因为膜蛋白是结构测定的优先目标，现有的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。首先确定具有两个跨膜螺旋的MerF和MerE的结构，然后对具有三个（MerT）和四个（MerC） TM螺旋的该家族其他成员进行研究。研究这一蛋白质家族在研究中的第二个作用是为固体核磁共振波谱的仪器和实验方法的发展提供越来越大和复杂的蛋白质靶标。这些蛋白之间的比较可能有助于了解为什么能够解毒汞的独立细菌分离物具有不同数量的转运蛋白，蛋白质具有不同数量的TM螺旋，以及蛋白质具有不同数量的结合汞的半胱氨酸残基对。第三个目标来自技术的发展和该蛋白家族成员的结构发现，这为汞转运膜蛋白的二元和三元复合物的组装和结构研究奠定了基础，这些复合物具有质周蛋白MerP（我们之前确定了其结构）和汞还原酶的n端“MerP样”结构域MerA（其结构已被其他人确定）。汞还原酶（MerA）将剧毒的汞（II）还原为毒性较小且易挥发的汞(0)，被动地扩散出细胞。将Hg（II）从外周质转运到细胞质是一个关键步骤，必须严格控制，使高活性的Hg（II）在溶液中永远不会游离，并可与必需细胞蛋白上的半胱氨酸残基反应，这是其在没有mer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修改，核磁共振实验的发展和执行，以及结构计算。汞转运膜蛋白的结构及其功能复合物为跨双层膜转运汞（II）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第一个例子之一，因为器官汞化合物在食物供应（特别是在大型鱼类中）和环境中广泛分布。