NMR STRUCTURAL STUDIES OF PRION PROTEINS WITH POINT MUTATIONS

尽管可以获得许多物种的细胞朊病毒蛋白的大量结构信息，但对于引起疾病的痒病形式的蛋白质，除了从 CD 光谱中获得的二级结构内容的基本知识之外，几乎没有什么可利用的。鉴于这种形式的蛋白质的多聚体和不溶性性质，如果样品制备或光谱技术没有突破，似乎不太可能获得这种形式的蛋白质的高分辨率结构信息。解决这个问题的一种方法是检查家族性朊病毒疾病中发现的蛋白质突变形式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，该项目将研究最小基因的三种突变形式的结构和动力学。 小鼠朊病毒蛋白的感染结构域 (PrP90-231)。在具体目标 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，与野生型相比，表现出较低的朊病毒病倾向）的突变蛋白的多肽链动力学。这些研究将对该计划具有直接的相关性和实用性，提供可用于基于结构的治疗设计的详细结构信息。控制系统，即遗传性 Gerstmann-Straussler-Scheinker 病中常见的 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解决方案结构，该蛋白显示出朊病毒形成的倾向增加。通过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 NMR 化学位移图技术对本计划中开发的治疗药物的结合位点进行可视化。具体目标 1-3 中的研究应证明可直接用于该计划项目的其他组成部分，提供对新颖、有效疗法的迭代设计至关重要的信息。此外，预计将获得关于朊病毒蛋白转化为不溶性致病形式的结构基础的重要见解。

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.