NMR STRUCTURAL STUDIES OF PRION PROTEINS WITH POINT MUTATIONS

虽然从许多物种获得了大量关于细胞型Pron蛋白的结构信息，但除了CD光谱中可获得的二级结构内容之外，几乎没有什么可用于致病的瘙痒病形式的蛋白质。鉴于这种蛋白质的多聚体和不溶性，在没有样品制备或光谱技术突破的情况下，关于这种蛋白质的高分辨率结构信息似乎不太可能出现。解决这个问题的一种方法是检查在家族性Pron疾病中发现的突变形式的蛋白质对疾病敏感性增强的潜在结构基础，以及某些显性负Prion蛋白突变体发现的敏感性降低的潜在结构基础。为了解决这个问题，这个项目将研究三种最小突变形式的结构和动力学 小鼠蛋白的感染区(PrP90-231)。在特定目标1中，将计算高分辨率核磁共振溶液结构，并将分析两个具有显性负表型(即，显示出比野生型更低的Pron病倾向)的突变蛋白质的多肽链动力学。这些研究将对该计划具有直接的相关性和实用性，提供详细的结构信息，可用于基于结构的治疗设计。一个控制系统，经常在遗传性Gerstmann-Straussler-Scheinker病中发现的P102L突变蛋白，将在特定目标2中进行研究。将计算该蛋白的高分辨率溶液结构，这表明Pron形成的倾向增加。核磁共振测量的多肽链动力学也将是该项目这一部分的重要组成部分。具体目标3涉及直接 使用核磁共振化学位移映射技术可视化本计划中开发的治疗药物的结合部位。具体目标1-3中的研究应该通过提供对新的、有效的治疗方法的迭代设计至关重要的信息，证明对计划项目的其他组成部分有直接的用处。此外，预计将对Pron蛋白转化为不容溶解的致病形式的结构基础获得重要见解。

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.