NMR Structure and Dynamics of Membrane Binding Proteins

描述(申请人提供)：本项目的总体目标是使用多维异核磁共振(核磁共振)波谱来 表征三维(3D)结构、膜结合 生物重要性的机制、多聚化界面和动力学 与人类疾病有关的蛋白质。每个域被预测为代表一个 新的结构折叠，并已被证明直接结合或折叠在上面 膜。我们研究的具体目标是： α-突触核蛋白：α-突触核蛋白是一种动态的140个残基蛋白，在酸性膜上折叠成螺旋结构，然后寡聚为 贝塔薄片原纤维。蛋白质被突变驱动进入后一种状态 与帕金森氏症有关。胶束结合和胶束结合的溶液结构 我们将阐明和比较α-突触核蛋白的游离态。该结构 连接酸性磷脂和金属的口袋将被定义为 在这个过程中，那些被磷酸化、亚硝化或截断的位点 聚合的过程。构象和动力学的变化 发生齐聚并折叠到胶束表面将是 调查过了。 甘氨酸受体：甘氨酸受体是一个超家族的原型成员 由甘氨酸、GABA调节的离子通道。5-羟色胺和烟碱型乙酰胆碱 神经递质。我们已经确定了甘氨酸的胞外结构域 受体通过有限的蛋白降解，并证明了它们的功能 正直。近100个残基神经递质的三维结构 该受体的结合结构域将通过核磁共振进行表征。的结构 激动剂、士的宁、磷脂、锌和多肽的结合部位 将定义到离子通道的信号。 海滩域：Chediak-Higashi综合征是一种潜在的致命人类遗传病，由CHSi蛋白及其海滩域突变引起 扰乱溶酶体的贩运。我们已经确定了这部小说的界限 大约150个残基结构域，并已表明它与脂类相关。 在这里，我们阐明了它的低聚状态和存在下的三维结构 胶束揭示膜相互作用和蛋白质的结构基础 承认。 DIX结构域：DIX结构域是一个新的约85个残基的信号模块，在Wnt信号通路中发挥关键作用 胚胎发育和癌症进展。该问题的解结构 蓬乱的和Axin DIX结构域的同源二聚体和单体将被阐明。这个 这些结构域的磷脂和蛋白质结合特性将是 以圆二色谱和荧光光谱为特征。这个 调节异二聚化和膜缔合的界面将是 经核磁共振、沉淀平衡和诱变实验鉴定 以定义这两个域的独特信号角色。

DESCRIPTION (provided by applicant): The overall aim of this project is to use multidimensional heteronuclear magnetic resonance (NMR) spectroscopy to characterize the three dimensional (3D) structures, membrane binding mechanisms, multimerization interfaces, and dynamics of biologically important proteins involved in human disease. Each domain is predicted to represent a novel structural fold and has been shown to bind directly to or fold on membranes. The specific targets of our study are: alpha-Synuclein: The alpha-synuclein protein is a dynamic 140 residue protein that folds into a helical structure on acidic membranes and oligomerizes into beta sheet fibrils. The protein is driven into the latter state by mutations linked to Parkinson's disease. The solution structures of the micelle-bound and free states of alpha-synuclein will be elucidated and compared. The structure of the pockets that ligate acidic phospholipids and metals will be defined, as will the sites that become phosphorylated, nitrosylated, or truncated during the course of polymerization. The changes in conformation and dynamics that occur upon oligomerization and folding onto micelle surfaces will be investigated. Glycine Receptor: The glycine receptor is a prototypic member of a superfamily of ion channels gated by glycine, GABA. serotonin, and nicotinic acetylcholine neurotransmitters. We have identified the extracellular domains of the glycine receptor by limited proteolysis and have demonstrated their functional integrity. The 3D structure of the approximately 100 residue neurotransmitter binding domain of this receptor will be characterized by NMR. The structure of the binding sites for agonists, strychnine, phospholipids, zinc, and peptides that signal to the ion channel will be defined. BEACH Domain: The Chediak-Higashi syndrome is a potentially fatal human genetic disorder caused by mutations in the CHSI protein and its BEACH domain that disrupt lysosomal trafficking. We have defined the boundaries of this novel approximately 150 residue domain and have shown that it associates with lipids. Here we elucidate its oligomeric state and 3D structure in the presence of micelles to reveal the structural basis of membrane interaction and protein recognition. DIX Domain: The DIX domain is a novel approximately 85 residue signaling module that plays a key role in the Wnt signaling pathway that contributes to embryonic development and cancer progression. The solution structures of Disheveled and Axin DIX domain homodimers and monomers will be elucidated. The phospholipid and protein binding properties of these domains will be characterized by circular dichroism and fluorescence spectroscopy. The interfaces that mediate heterodimerization and membrane association will be identified by NMR, sedimentation equilibrium, and mutagenesis experiments in order to define the unique signaling roles of these two domains.