PRP STRUCTURE AND POLVOXOMETALATES

项目主任/首席调查员(最后、第一、中间)：普鲁塞纳、斯坦利/朗、杰弗里(项目1) 项目总结(见说明)： 磷钨酸盐阴离子(PTA)与传染性蛋白亚型(PrPSc)特异结合，但 而不是它的细胞前体(PrPC)。虽然PTA现在被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的四元结构及其N-末端截断形式PrP 27- 30.PTA是一种多金属氧酸盐(POM)，是一种具有刚性多面体结构的无机金属氧化物簇合物。 POMS是一大类化合物，在化学成分、大小、 形状和电荷密度。我们假设POMS和PrPSc之间的结合是可以操纵的 通过改变POM的化学成分，从而影响其大小、形状和电荷。按顺序 为了深入了解POM-PrPSc相互作用的构象选择性，我们计划研究 通过核磁共振和红外光谱对结合的多金属有机化合物的结构、化学计量和形态进行了研究。在……里面 此外，我们计划使用POMS作为工具来修改PrP50的聚合属性，目的是 生产用于结构分析的更高质量的二维晶体和淀粉原纤维。第四纪 将用电子显微镜和扫描探针分析POM-Prion聚集体的结构 显微镜。相反，一些较大的POM不能沉淀PrPSc，而是解离PrPSc聚集体。 这一意想不到的特性将被利用来开发具有传染性的天然PrPSc的增溶方案。 不同的生物物理性质将被用作增溶标准。生物检测将被广泛应用于 测试溶解形式的PrPSc是否仍具有传染性。一种可溶性均一的PrPSc制备方法 ^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分析 等温滴定热法(ITC)。ITC允许测定化学计量比、结合常数(公斤)、 反应热(AH)和标准结合自由能(AG)。 相关性(请参阅说明)： 确定PrPSc？POM络合物形成的反应参数应有助于结构 了解POMS和PrPSc之间的相互作用，帮助选择具有有利条件的新POMS 属性，并使关于合成其他POM的更合理的决策。到目前为止，相对较少的 探索了以钨和钼为基础，具有选定的杂原子的多金属有机化合物。种类繁多 多金属有机化合物的种类仍有待测试，包括以钒和铌为基础的水溶性多金属有机化合物。 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/