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

描述(申请人提供)：扩散性淀粉样蛋白中间体的分子结构，通常在淀粉样蛋白错误折叠成纤维中观察到，引起了广泛的兴趣，因为这些中间体是强大的神经毒素，可能与阿尔茨海默病(AD)等淀粉样疾病有关，并且因为中间体的结构将为错误折叠途径提供新的见解。然而，由于中间体本质上的不稳定性、非均质性和非结晶性，传统的方法，如X射线结晶学和溶液核磁共振，已经不能有效地阐明它们的分子结构。这项研究的长期目标是用固体核磁共振(SS核磁共振)来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结构测定的潜在问题有两个：(1)缺乏有效的方案来分离特定的瞬时中间体；(2)缺乏确定位点分辨结构的方法。 我们已经开发了一种使用SS核磁共振的新方法，该方法将允许对40个残基的阿尔茨海默病β-淀粉样多肽Abeta(1-40)的纤维形成中的中间产物进行现场解析结构测定。在这种方法中，通过光学光谱和电子显微镜(EM)检测淀粉样肽的构象和形态变化。然后，利用SS核磁共振对冻结捕获的中间产物进行了定量结构检查，这已经成为分析热稳定的淀粉样纤维结构的主要方法。我们假设，在Abeta(1-40)的错误折叠中，含有β-折叠的可扩散中间体存在于光化之前。基于这一假设，我们将在低温下使用EM和荧光检测可溶的β-折叠中间体，这减缓了纤维的形成，并在硫代黄素T(Tht)染料的存在下检测到，硫代黄素T(Tht)染料是富含β-折叠的聚集体的指示剂。错误折叠动力学将进一步通过对EM图像、CD光谱和THT荧光的孵化-时间依赖性分析来检验。然后，我们将通过各种SS核磁共振方法确定Abeta(1-40)的β-折叠中间体的位置分辨二级结构和超分子结构，该中间体被冷冻捕获并随后冷冻干燥。这些测量将检验我们的假设，即在Abeta(1-40)纤化之前存在直径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E22G突变体的动力学和位置分辨中间结构，Abeta(1-40)已知可以稳定亚纤维中间体。对于这个突变体，我们将分离出两个直径约为20 nm和20-30 nm的不溶球形中间体，这两个中间体都含有β-折叠结构。E22G中间体中的β-折叠区域的位置将通过SS核磁共振进一步确定。

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.