STRUCTURAL STUDIES OF PRION CONFORMATIONAL CONVERSION

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。这项提议是继续使用小角X射线散射(SAXS)对由于从α-螺旋形式到丰富的β-形式的构象转换而自我结合的少聚型普恩蛋白的结构研究。以前的研究已经为哺乳动物重组PrP的六聚体形式建立了一个“三聚体”原子尺度模型，可以直接与直接从动物大脑中获得的阴染的刮痕状蛋白质的电子显微镜数据进行比较。这种比较允许对非感染性重组形式和感染性疾病形式之间的差异进行结构界限。我们计划通过使用CCD探测器获得更高分辨率的数据来扩大这些研究，并将研究扩展到从酵母中提取的一种蛋白质Sup-35，这种蛋白质具有类似Pron的特性。通过光散射、电子显微镜和原子力显微镜的研究，证实了低聚中间体存在于折叠过程中。我们还计划测量蛋白质片段srcSH3的低聚形式，我们有一系列丙氨酸替代突变体。根据Dobson和他的合作者的工作，他们已经证明淀粉样纤维的形成似乎是多肽自结合的普遍现象，我们已经证明这种蛋白质在滞后阶段后形成纤维。研究寡聚体的形成依赖于点突变，应该有助于验证蛋白质自关联的结构模型，如结构域交换，或为理解这一基本现象提供新的方向，这在阿尔茨海默氏症和帕金森氏症等疾病中具有重要意义。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This proposal is to continue structural studies using small angle x-ray scattering (SAXS) on oligomeric forms of prion proteins which self associate as a result of conformational conversion from an alpha-helical form to a beta-rich form. Previous studies have led to the formulation of a "trimer-of-dimers" atomic scale model for a hexameric form of the mammalian recombinant PrP which can be compared directly with electron microscope data from negatively stained samples of the scrapie form of the protein obtained directly form animal brains. This comparison allows structural bounds to be placed on the differences between the non-infective recombinant form and the infective disease form. We plan to extend these studies by obtaining higher resolution data with the use of the CCD detector, and also to extend the studies to a protein , Sup-35, from yeast which has prion-like properties. and from which the existence of oligomeric intermediates along the folding pathway has been established by light scattering, em and afm work. We also plan to measure oligomeric forms of a protein fragment srcSH3 for which we have a series of alanine-replacement mutants. Following work of Dobson and collaborators, who have demonstrated that formation of amyloid fibrils appears to be a universal for self-association of polypeptides, we have shown that this protein forms fibrils after a lag phase. Study of the dependence of the formation of oligomers on point mutations, should help in validation of structural models for protein self-association such as domain swapping, or suggest new directions for understanding this fundamental phenomenon which is of importance in diseases such as Alzheimer's and Parkinson's.