NMR STRUCTURAL STUDIES OF PRION PROTEINS WITH POINT MUTATIONS

虽然从许多物种的细胞朊病毒蛋白中获得了大量的结构信息，但除了从CD光谱中获得的二级结构含量的知识之外，很少有关于该蛋白的致病痒病形式的知识。考虑到这种形式的蛋白质的多聚性和不溶性，在没有样品制备或光谱技术突破的情况下，似乎不太可能获得这种形式蛋白质的高分辨率结构信息。解决这一问题的一种方法是研究家族性朊病毒疾病中发现的蛋白质突变形式的疾病易感性增强的潜在结构基础，以及某些显性阴性朊病毒蛋白质突变体的易感性降低。为了解决这个问题，这个项目将研究三种突变形式的最小体的结构和动力学

Although a large amount of structural information is available on the cellular prion protein from a number of species, little beyond the bare knowledge of secondary structure content available from CD spectra is available for the disease-causing scrapie form of the protein. Given the multimeric and insoluble nature of this form of the protein, it appears unlikely that high-resolution structural information will be forthcoming on this form of the protein in the absence of breakthroughs in sample preparation or spectroscopic techniques. One approach to this problem is to examine the underlying structural basis for the enhanced disease susceptibility of mutant forms of the protein found in familial prion diseases, together with the reduced susceptibility found for certain dominant negative prion protein mutants. In order to address this question, this project will investigate the structure and dynamics of three mutant forms of the minimal infective domain (PrP90-231) of the mouse prion protein. In Specific Aim 1, the high-resolution NMR solution structures will be calculated and polypeptide chain dynamics will be analyzed for two mutant proteins that exhibit dominant negative phenotypes (that is, exhibit a lower propensity for prion disease than wild-type). These studies will have direct relevance and utility for the Program, providing detailed structural information that can be used for structure-based design of therapeutics. A control system, the P102L mutant protein frequently found in the inherited Gerstmann-Straussler-Scheinker disease, will be studied in Specific Aim 2. A high-resolution solution structure will be calculated for this protein, which shows increased propensity for prion formation. Polypeptide chain dynamics measured by NMR will also be an important component of this part of the project. Specific Aim 3 involves the direct visualization of the sites of binding of the therapeutic drugs developed in this Program using the technique of NMR chemical shift mapping. The studies in Specific Aims 1-3 should prove to be of direct use to other components of the Program Project, by providing information vital to the iterative design of novel, effective therapeutics. In addition, it is anticipated that important insights will be gained into the structural basis of the conversion of prion proteins to insoluble disease-causing forms.