PRP STRUCTURE AND POLVOXOMETALATES

项目主任/主要研究者（最后，第一，中间）：Prusiner，Stanley/Long，Jeffrey（项目1） 项目总结（见说明）： 磷钨酸盐阴离子（PTA）特异性结合朊病毒蛋白（PrPSc）的感染性同种型，但 而不是其细胞前体（PrPc）。虽然PTA现在被广泛用于沉淀和纯化PrPSc， 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是聚氧乙烯酸盐（POM），一种具有刚性多面体结构的无机金属氧化物簇。 POM是在化学组成、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的化学组成，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 结构，化学计量，并通过核磁共振和红外光谱结合的POM形态。在 此外，我们计划使用POM作为工具来修改PrP50的聚集特性，目的是 产生更高质量的二维晶体和淀粉样纤维用于结构分析。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不能沉淀PrPSc，而是解离PrPSc聚集体。 将利用这一意想不到的特性来开发感染性天然PrPSc的增溶方案。 不同的生物物理性质将用作增溶标准。生物测定将被广泛用于 测试PrPSc的溶解形式是否保持感染性。一种可溶性均质的PrPSc制剂 ^C 可以进行大量的结构研究。将通过以下方法分析各种POM对PrP的亲和力： 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分析。 了解POM和PrPSc之间的相互作用，有助于选择具有有利的 的性质，并使更多的合理的决定合成额外的POM。到目前为止， 已经探索了基于具有选定杂原子的钨和钼的POM。各种各样 的POM物种仍有待测试，包括基于钒和铌的水溶性POM。 项目/

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/