HYDROGEN EXCHANGE STUDIES ON A-BETA AMYLOID FIBRILS

描述(摘自申请者摘要)：发现淀粉样纤维 与越来越多的人类疾病有关，包括几种 神经退行性疾病，其中最常见的是阿尔茨海默病 (Ad)。在每一种疾病中，作为主要成分的蛋白质 淀粉样原纤维是不同的。因此，淀粉样蛋白是一种特征性的 聚集结构，而不是特定的蛋白质分子。事实上，在那里 是否有人推测这种淀粉样蛋白聚集体结构可能是一种 可以被许多蛋白质序列访问的蛋白质折叠基序 合适的环境。因为它与人类疾病和 对蛋白质折叠的基本了解，尤其重要的是 更详细地了解淀粉样原纤维的折叠结构。淀粉样原纤维 不适用于测定蛋白质的标准技术 结构，因此我们对此结构了解甚少。 分子水平超越了这些聚集体富含β-折叠的事实 二级结构。我们选择研究与阿尔茨海默病相关的淀粉样蛋白， 由A-β多肽组成。在这里提出的研究中，我们将使用 氢-氚交换(HX)技术绘制二级结构图 原纤维中的A-β。氢键，如在多肽之间发现的氢键 β片中的主链酰胺氢，保护酰胺氢不被交换 而大多数其他酰胺氢是自由交换的。这样做的具体目的是 应用是(1)开发收集A-beta上的HX数据的方法 结合成淀粉样纤维，使用质谱仪(MS)和 核磁共振(核磁共振)来量化交换水平，并使用这些 方法定位受保护的和暴露的A-β的酰胺氢 纤维；(2)对其他A-β聚集体进行类似的交换研究，如 原纤维，与淀粉样蛋白组装和Abeta有关 聚合毒性；(3)结合HX数据使用计算方法 建立、测试和改进原丝和原纤维结构的模型；以及 (4)对其他A-β聚集体进行交换实验，包括 由A-β片段和神经炎斑块核心在体外制成的纤维 从人类阿尔茨海默病脑材料中分离出来。这些研究将使我们更仔细地了解 淀粉样原纤维的结构，这将使我们能够更好地 了解纤维是如何生长的，以及它们是如何破坏人类细胞和组织的。一个 结构知识的提高也将提高我们识别和识别 设计抑制A-β生长和毒性的治疗分子 淀粉样蛋白和其他聚集体。

DESCRIPTION (From the Applicant's Abstract): Amyloid fibrils are found associated with a growing number of human diseases, including several neurodegenerative diseases, the most prevalent of which is Alzheimer's Disease (AD). In each of these diseases, the protein that is the main component of the amyloid fibril is different. Thus, amyloid is a characteristic type of aggregate structure, rather than a particular protein molecule. Indeed, there is some speculation that this amyloid type of aggregate structure may be a protein folding motif that can be accessed by many protein sequences under the right circumstances. Because of its relevance to both human disease and fundamental understanding of protein folding, it is particularly important to know in more detail the folded structure of the amyloid fibril. Amyloid fibrils do not lend themselves to standard techniques for determining protein structure, with the result that we know very little about this structure at the molecular level beyond the fact that these aggregates are rich in beta sheet secondary structure. We have chosen to work on the amyloid associated with AD, composed of the peptide A-beta. In the research proposed here we will use the technique of hydrogen-deuterium exchange (HX) to map the secondary structure of A-beta in the fibril. Hydrogen bonds, such as are found between polypeptide backbone amide hydrogens in beta sheet, protect amide hydrogens from exchange while most other amide hydrogens freely exchange. The specific aims of this application are to (1) Develop methods for collecting HX data on A-beta incorporated into amyloid fibrils, using both mass spectrometry (MS) and nuclear magnetic resonance (NMR) to quantify levels of exchange, and use these methods to map the protected and exposed amide hydrogens of A-beta in the fibril; (2) carry out similar exchange studies on other A-beta aggregates, like protofibrils, that are implicated in both amyloid assembly and in Abeta aggregate toxicity; (3) use computational methods in concert with the HX data to build, test, and refine models of protofilament and fibril structure; and (4) conduct exchange experiments on other aggregates of A-beta, including fibrils made in vitro from A-beta fragments as well as neuritic plaque cores isolated from human AD brain material. These studies will give us a closer look at the structure of the amyloid fibril, which will allow us to better understand how fibrils grow and how they disrupt human cells and tissue. An improved knowledge of structure will also improved our ability to identify and design therapeutic molecules for inhibiting the growth and toxicity of A-beta amyloid and other aggregates.