STRUCTURAL STUDIES OF PRION CONFORMATIONAL CONVERSION

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。该建议是继续使用小角度x射线散射（SAXS）对朊病毒蛋白的低聚形式进行结构研究，这些蛋白由于从α -螺旋形式到β -富形式的构象转换而自结合。先前的研究已经为哺乳动物重组PrP的六聚体形式建立了“三聚体-二聚体”原子尺度模型，可以直接与直接从动物大脑中获得的痒病形式蛋白质的阴性染色样品的电子显微镜数据进行比较。这种比较允许对非传染性重组形式和传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展这些研究，并将研究扩展到酵母中具有朊病毒样特性的蛋白质su -35。并通过光散射、em和afm功确定了折叠路径上存在低聚中间体。我们还计划测量蛋白质片段srcSH3的低聚形式，我们已经有了一系列的丙氨酸替代突变体。Dobson和他的合作者已经证明淀粉样蛋白原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期形成原纤维。研究低聚物的形成对点突变的依赖性，应该有助于验证蛋白质自关联的结构模型，如结构域交换，或为理解这一在阿尔茨海默氏症和帕金森病等疾病中重要的基本现象提供新的方向。

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.