NMR STRUCTURAL STUDIES OF PRION PROTEINS WITH POINT MUTATIONS

虽然大量的结构信息是从一些物种的细胞朊病毒蛋白，除了从CD光谱的二级结构内容的裸露知识是可用于引起疾病的瘙痒症形式的蛋白质。鉴于这种形式的蛋白质的多聚体和不溶性的性质，在样品制备或光谱技术没有突破的情况下，这种形式的蛋白质似乎不太可能获得高分辨率的结构信息。解决这个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，以及某些显性负性朊病毒蛋白突变体的易感性降低。为了解决这个问题，本项目将研究三种突变形式的最小的结构和动力学 小鼠朊病毒蛋白的感染性结构域（PrP 90 -231）。在特定目标1中，将计算高分辨率NMR溶液结构，并分析两种表现出显性负表型（即，表现出比野生型更低的朊病毒疾病倾向）的突变蛋白的多肽链动力学。这些研究将与该计划直接相关和实用，提供详细的结构信息，可用于基于结构的治疗设计。控制系统，P102 L突变蛋白经常发现的遗传性Gerstmann-Straussler-Scheinker病，将在具体目标2研究。将计算该蛋白质的高分辨率溶液结构，其显示朊病毒形成的倾向增加。通过NMR测量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NMR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位点。具体目标1-3中的研究应通过提供对新型有效治疗方法的迭代设计至关重要的信息，证明对项目的其他组成部分具有直接用途。此外，预计重要的见解将获得朊病毒蛋白转化为不溶性致病形式的结构基础。

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.