PRP STRUCTURE AND POLVOXOMETALATES

PRP 结构和多氧金属盐

• 批准号: 8374611
• 负责人: Jeffrey Long
• 金额: $ 21.71万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8374611
• 关键词: Affinity; Aging; Anions; Binding; Biological Assay; Calorimetry; Charge; Chemicals; Complex; Disease; Electron Microscopy; Family; Free Energy; Instruction; Molecular; Molybdenum; NMR Spectroscopy; Niobium; PrP; PrPSc Proteins; Preparation; Principal Investigator; Prions; Property; Protein Isoforms; Protocols documentation; Reaction; Scanning Probe Microscopy; Shapes; Structure; Testing; Titrations; Tungsten; Vanadium; Variant; Water; base; density; enthalpy; infrared spectroscopy; insight; metal oxide; programs; stoichiometry; tool; two-dimensional

Program Director/Principal Investigator (Last, First, Middle): Prusiner, Stanley/Long, Jeffrey (Project 1) PROJECT SUMMARY (See instructions): The phosphotungstate anion (PTA) binds specifically to the infectious isoform of the prion protein (PrPSc), but not to its cellular precursor (PrPc). Although PTA is now widely used to precipitate and purify PrPSc, the molecular details and the mechanism of this interaction are not understood. We have observed that the interaction with PTA influences the quaternary structure of PrPSc and its N-terminally truncated form, PrP 27- 30. PTA is.a polyoxometalate (POM), an inorganic metal oxide cluster with a rigid polyhedral structure. POMs are a large family of compounds displaying substantial variations in chemical composition, size, shape, and charge density. We hypothesize that the binding between POMs and PrPSc can be manipulated by changing the chemical composition of the POM, thereby influencing its size, shape, and charge. In order to gain insights into the conformational selectivity of the POM-PrPSc interaction, we plan to investigate the structure, stoichiometry, and speciation of the bound POMs through NMR and infrared spectroscopy. In addition, we plan to use POMs as tools to modify the aggregation properties of PrP50 with the aim of producing higher-quality two-dimensional crystals and amyloidfibrils for structural analyses. The quaternary structure of the POM-prion aggregates will be analyzed by electron microscopy and scanning probe microscopy. In contrast, some larger POMs fail to precipitate PrPSc, and instead dissociate PrPSc aggregates. This unexpected property will be exploited to develop a solubilization protocol for infectious, native PrPSc. Different biophysical properties will be used as solubilization criteria. Bioassays will be utilized extensively to test whether solubilized forms of PrPSc remain infectious. A soluble and homogeneous preparation of PrPSc ^ c would enable numerous structural studies. The affinity of the various POMs for PrP will be analyzed by isothermal titration calorimetry (ITC). ITC allows determination of the stoichiometry, binding constant(Kg), reaction enthalpy (AH), and the standard free energy of binding (AG). RELEVANCE (See instructions): Determining the reaction parameters for the PrPSc¿POM complex formation should facilitate the structural understanding of the interaction between POMs and PrPSc, aid in the selection of new POMs with favorable properties, and enable more rational decisions about the synthesis of additional POMs. So far, relatively few POMs, based on tungsten and molybdenum with selected heteroatoms, have been explored. A wide variety of POM species remain to be tested, including water-soluble POMs based upon vanadium and niobium. PROJECT/

项目总监/首席研究员（最后、第一、中间）：Prusiner, Stanley/Long, Jeffrey（项目 1） 项目摘要（参见说明）： 磷钨酸阴离子 (PTA) 特异性结合朊病毒蛋白 (PrPSc) 的传染性亚型，但 不与其细胞前体 (PrPc) 相关。虽然 PTA 现在广泛用于沉淀和纯化 PrPSc，但 这种相互作用的分子细节和机制尚不清楚。我们观察到 与 PTA 的相互作用影响 PrPSc 的四级结构及其 N 末端截短形式 PrP 27- 30. PTA 是一种多金属氧酸盐（POM），一种具有刚性多面体结构的无机金属氧化物簇。 POM 是一大类化合物，在化学成分、尺寸、 形状和电荷密度。我们假设 POM 和 PrPSc 之间的结合可以被操纵 通过改变 POM 的化学成分，从而影响其尺寸、形状和电荷。为了 为了深入了解 POM-PrPSc 相互作用的构象选择性，我们计划研究 通过 NMR 和红外光谱分析结合的 POM 的结构、化学计量和形态。在 此外，我们计划使用 POM 作为工具来修改 PrP50 的聚合属性，目的是 生产更高质量的二维晶体和淀粉样原纤维用于结构分析。第四纪 POM-朊病毒聚集体的结构将通过电子显微镜和扫描探针进行分析 显微镜。相反，一些较大的 POM 无法沉淀 PrPSc，而是解离 PrPSc 聚集体。 这种意想不到的特性将被用来开发传染性天然 PrPSc 的溶解方案。 不同的生物物理性质将被用作溶解标准。生物测定法将被广泛用于 测试溶解形式的 PrPSc 是否仍具有传染性。 PrPSc 的可溶性均质制剂 ^c 将使大量的结构研究成为可能。各种 POM 对 PrP 的亲和力将通过以下方式进行分析： 等温滴定量热法（ITC）。 ITC 允许测定化学计量、结合常数（Kg）、 反应焓 (AH) 和标准结合自由能 (AG)。 相关性（参见说明）： 确定 PrPSc¿POM 复合物形成的反应参数应有助于结构 了解 POM 和 PrPSc 之间的相互作用，有助于选择具有有利特性的新 POM 性质，并使有关额外 POM 合成的更合理决策成为可能。到目前为止，相对较少 基于具有选定杂原子的钨和钼的 POM 已得到探索。种类繁多 多种聚甲醛有待测试，包括基于钒和铌的水溶性聚甲醛。 项目/