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: 7278155
• 负责人: YOSHITAKA ISHII
• 金额: $ 26.72万
• 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/7278155
• 关键词: Alzheimer' s Disease; Amyloid; Amyloid Fibrils; Amyloid Proteins; Amyloid beta-Protein; Amyloidosis; Arctic Regions; C-terminal; Caliber; Cell Death; Chemicals; Circular Dichroism; Data; Dependence; Dyes; Electron Microscopy; Fluorescence; Fluorescence Microscopy; Fluorescence Spectroscopy; Freeze Drying; Freezing; Heterogeneity; Image; Kinetics; Location; Measurement; Measures; Methods; Modeling; Molecular; Molecular Conformation; Molecular Structure; Monitor; Morphology; Mutation; Nature; Neurons; Neurotoxins; Optics; Pathway interactions; Peptides; Protocols documentation; Range; Research; Research Personnel; Resolution; Sampling; Senile Plaques; Site; Solid; Solutions; Spectrum Analysis; Structure; Temperature; Testing; Thioflavin T; Time; Water; X-Ray Crystallography; amyloid peptide; base; beta pleated sheet; cold temperature; insight; interest; mutant; neurotoxic; neurotoxicity; novel strategies; physical separation; programs; research study; solid state; therapeutic target

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.

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.然而，由于中间体的本质不稳定，异质和非结晶性，传统方法（例如X射线晶体学和溶液NMR）无法阐明其分子结构。这项研究的长期目的是确定固态NMR（SSNMR）将淀粉样蛋白折叠成原纤维的结构变化。淀粉样蛋白中间体的结构确定的基本问题是双重的（1）缺乏隔离特定瞬态中间体的有效方案，以及（2）缺乏确定现场分辨结构的方法。 我们已经开发了一种使用SSNMR的新方法，该方法将允许在40个沉重的阿尔茨海默氏症β-淀粉样蛋白肽Abeta（1-40）的原纤维形成中的现场分辨结构测定。在这种方法中，通过光谱和电子显微镜（EM）检测淀粉样肽的构象和形态变化。然后，通过SSNMR对冻伤的中间体进行定量结构检查，这是对热稳定淀粉样蛋白原纤维结构分析的主要方法。我们假设在纤维化之前存在一个可扩散的中间体的ABETA（1-40）中折叠时（1-40）。基于该假设，我们将在降低温度下检测可溶性β-折叠中间体，该中间体在存在硫牛粉T（THT）染料的情况下使用EM和荧光放慢了原纤维的形成，这是β-片蛋白含量富的聚集体的指标。通过分析EM图像，CD光谱和THT荧光的孵育时间依赖性，将进一步研究错误折叠的动力学。然后，我们将确定位置分辨的二级结构和Abetaβ-片中间体的超分子结构（1-40），该中级（1-40）被各种SSNMR方法冻结并随后冻干。这些测量结果将检验我们的假设，即直径为15-30 nm的球形淀粉样蛋白中间体存在于Abeta（1-40）（1-40）之前，并且中间体在C-末端和疏水性核心中包含有序的β-beta表。我们还将研究Abeta的E22G突变体（1-40）的动力学和位置分辨中间结构，该突变体已知可以稳定亚纤维中间体。对于这个突变体，我们将在大约20 nm和20-30 nm的直径中分离两个不溶性球形中间体，它们都包含β-片结构。 E22G中间体中β-片区域的位置将由SSNMR进一步识别。