Molecular Structures and Kinetics of Amyloid Intermediates by Solid-State NMR

通过固态核磁共振研究淀粉样蛋白中间体的分子结构和动力学

• 批准号: 7131493
• 负责人: YOSHITAKA ISHII
• 金额: $ 24.69万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7131493
• 关键词: amyloid proteins; bioimaging /biomedical imaging; chemical kinetics; electron microscopy; flavins; fluorescence microscopy; fluorescent dye /probe; molecular dynamics; mutant; neurofibrillary tangles; nuclear magnetic resonance spectroscopy; pathologic process; protein folding; protein metabolism; protein structure; structural biology; thermostability; thiols

DESCRIPTION (provided by applicant): Molecular structures of diffusible amyloid intermediates, commonly observed in misfolding of amyloid proteins into fibrils, have attracted broad interest because these intermediates are potent neurotoxins that may be responsible for amyloid diseases such as Alzheimer's disease (AD) and because structures of the intermediates would provide new insight into the misfolding pathway. However, owing to the intrinsically unstable, heterogeneous, and non-crystalline nature of the intermediates, traditional approaches, such as X-ray crystallography and solution NMR, have been ineffective for elucidating their molecular structures. The long-term objective of this research is to determine the structural changes of amyloid proteins in the course of misfolding into fibrils by solid-state NMR (SSNMR). The underlying problems hindering structural determination of amyloid intermediates are twofold (1) lack of effective protocols to isolate a specific transient intermediate, and (2) lack of methods to determine site-resolved structures. We have developed a novel approach using SSNMR that will permit the site-resolved structural determination of intermediates in fibril formation for a 40-residue Alzheimer's beta-amyloid peptide, Abeta(1-40). In this approach, conformational and morphological changes of the amyloid peptide are detected by optical spectroscopy and electron microscopy (EM). Then, quantitative structural examination is performed for freeze-trapped intermediates by SSNMR, which has been a dominant method for structural analysis of thermally stable amyloid fibrils. We hypothesize that in misfolding of Abeta(1-40), a diffusible intermediate containing beta-sheets exists prior to fibrillization. Based on the hypothesis, we will detect the soluble beta-sheet intermediates at reduced temperature, which slows fibril formations, using EM and fluorescence in the presence of a thioflavin T (ThT) dye, an indicator of beta-sheet-rich aggregates. The misfolding kinetics will be further examined by analysis of incubation-time dependence of EM images, CD spectra, and ThT fluorescence. Then, we will determine site-resolved secondary structures and supramolecular structures for the beta-sheet intermediate of Abeta(1-40), which is freeze trapped and subsequently lyophilized, by various SSNMR methods. These measurements will test our hypothesis that a spherical amyloid intermediate of 15-30 nm in diameter exists prior to fibrillization of Abeta(1-40) and that the intermediate contains well-ordered beta-sheets in the C-terminal and hydrophobic core. We will also examine kinetics and site-resolved intermediate structures for an E22G mutant of Abeta(1-40), which is known to stabilize subfibrillar intermediates. For this mutant, we will isolate two insoluble spherical intermediates in diameters of approximately 20 nm and 20-30 nm, both of which contain beta-sheet structures. The locations of the beta-sheet regions in the E22G intermediates will be further identified by SSNMR.

描述（由申请人提供）：在淀粉样蛋白错误折叠成原纤维中通常观察到的可扩散淀粉样中间体的分子结构引起了广泛的兴趣，因为这些中间体是可能导致淀粉样疾病（如阿尔茨海默病（AD））的强效神经毒素，并且因为中间体的结构将提供对错误折叠途径的新见解。然而，由于中间体的本质上不稳定，异质性和非结晶性，传统的方法，如X射线晶体学和溶液NMR，已经无法阐明其分子结构。本研究的长期目标是通过固体核磁共振（SSNMR）确定淀粉样蛋白在错误折叠成纤维过程中的结构变化。阻碍淀粉样蛋白中间体的结构测定的潜在问题是双重的（1）缺乏分离特定瞬时中间体的有效方案，和（2）缺乏测定位点分辨结构的方法。 我们已经开发了一种新的方法，使用SSNMR，将允许在原纤维形成的中间体的40个残基的阿尔茨海默氏β-淀粉样肽，Abeta（1-40）的位点分辨结构测定。在这种方法中，淀粉样肽的构象和形态变化通过光学光谱和电子显微镜（EM）检测。然后，通过SSNMR对冷冻捕获的中间体进行定量结构检查，SSNMR是热稳定淀粉样蛋白原纤维结构分析的主要方法。我们假设在Abeta（1-40）的错误折叠中，在折叠之前存在含有β折叠的可扩散中间体。基于这一假设，我们将在降低的温度下检测可溶性β-折叠中间体，这减缓了原纤维的形成，使用EM和荧光，在存在硫磺素T（ThT）染料的情况下，一种富含β-折叠的聚集体的指示剂。通过EM图像、CD光谱和ThT荧光的孵育时间依赖性分析，将进一步检查错误折叠动力学。然后，我们将通过各种SSNMR方法确定Abeta（1-40）的β折叠中间体的位点分辨二级结构和超分子结构，该中间体被冷冻捕获并随后冻干。这些测量将检验我们的假设，即在Abeta（1-40）的纤维化之前存在直径为15-30 nm的球形淀粉样蛋白中间体，并且该中间体在C-末端和疏水核心中含有有序的β-折叠。我们还将研究Abeta（1-40）的E22 G突变体的动力学和位点分辨中间体结构，已知该突变体可稳定亚原纤维中间体。对于该突变体，我们将分离两种直径约为20 nm和20-30 nm的不溶性球形中间体，这两种中间体均含有β-折叠结构。将通过SSNMR进一步鉴定E22 G中间体中β折叠区域的位置。