Raman Spectroscopic Studies of Amyloids

淀粉样蛋白的拉曼光谱研究

基本信息

批准号：
10008834
负责人：
Jennifer Lee
金额：
$ 31.71万
依托单位：
NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10008834
关键词：
Adopted Alzheimer&apos s Disease Amides Amyloid Amyloid Fibrils Amyloid Proteins Amyloidosis C-terminal Cells Characteristics Chemicals Coupled Deposition Detection Disease Environment Exhibits Extracellular Space Filament Fluorescence Frequencies Human Hydrogen Bonding Intervention Isotopes Label Lewy Bodies Ligation Methods Microscope Microscopic Microscopy Molecular Molecular Conformation Molecular Target N-terminal Nature Neurofibrillary Tangles Organ Parkinson Disease Pathogenicity Pattern Peptide Hydrolases Peptides Polymorph Positioning Attribute Protein Conformation Proteins Raman Spectrum Analysis Reporting Resistance Running Secondary Protein Structure Site Specificity Stretching Structure Techniques Tissues Vertebral column Width Work alpha helix alpha synuclein amyloid fibril formation amyloid formation beta pleated sheet insight interest novel polypeptide protein aggregation spectroscopic survey vibration

项目摘要

We have used Raman microspectroscopy to characterize amyloid formation of alpha-synuclein, which is implicated in Parkinsons disease. This direct spectroscopic method reports on intrinsic molecular vibrations such as protein amide bonds, which arise from coupled vibrational modes of the polypeptide backbone. The position and widths of the amide bands depend on the peptide-bond angles and hydrogen-bonding patterns, and therefore, inform on protein secondary structure as well as local environment. Conformations of alpha-helix, beta-sheet, or random coil exhibit characteristic peak maxima, making quantification of structural compositions possible. Specifically, we have coupled the Raman spectrometer with an inverted microscope, which yields both chemical and spatial information within macroscopic amyloid aggregates. Despite its high chemical specificity, Raman spectroscopy on its own does not offer site-specific information that is afforded by fluorescence and nitroxide-spin probes. To overcome this limitation, we have used native chemical ligation to produce segmentally 13C-labeled alpha-synuclein to provide region-specific structural details. As the 13C-isotope shifts the amide-I stretching frequency to lower energy, the ligated construct exhibits two distinct bands allowing for simultaneous detection of secondary structural changes in N-terminal 1-86 and C-terminal 87-140 residues during amyloid fibril formation. Interestingly, the formation of insoluble beta-sheet aggregates precedes filament formation (as observed by TEM) and enhanced thioflavin-T emission. Beta-sheet structure first forms in the N-terminal region, followed by the C-terminal region of ligated-alpha-synuclein. Further, we see that within a single, microscopic aggregate, the C-terminal region is less uniform than the N-terminal region. From this work, we gained both molecular and mechanistic insights into alpha-synuclein amyloid formation and we anticipate this strategy will be broadly applicable to other studies of amyloid formation.

我们已经使用拉曼微光谱法来表征α-核蛋白的淀粉样蛋白形成，这与帕金森氏病有关。这种直接的光谱法报告了固有分子振动（例如蛋白酰胺键），这些蛋白质是由多肽骨架的耦合振动模式引起的。酰胺带的位置和宽度取决于肽键角和氢键模式，因此，请告知蛋白质二级结构以及局部环境。 α-螺旋，β-片或随机线圈的构象表现出特征性峰值最大值，从而量化结构组合物。具体而言，我们将拉曼光谱仪与倒立显微镜耦合，该显微镜在宏观淀粉样蛋白聚集体中产生化学和空间信息。尽管具有很高的化学特异性，但拉曼光谱本身并未提供荧光和硝基氧化物旋转探针提供的特定地点信息。为了克服这一局限性，我们已经使用天然化学结扎术来产生分段标记的α-突触核蛋白，以提供特定区域的结构细节。随着13C同位素将酰胺-I拉伸频率转移到较低的能量时，绑扎的构建体表现出两个不同的频带，允许在淀粉样纤维纤维形成过程中同时检测N端1-86和C端87-140残基的二级结构变化。有趣的是，不溶性β-片骨料的形成先于细丝形成（如TEM所观察到的）和增强的硫洛鸟-T发射。 β-片结构在N末端区域首次形成，其次是连接的α-核蛋白的C末端区域。此外，我们看到在单个显微镜骨料中，C末端区域不如N末端区域均匀。从这项工作中，我们获得了分子和机械性见解，对α-突触核蛋白淀粉样蛋白形成，我们预计该策略将广泛适用于其他淀粉样蛋白形成研究。