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Solid State NMR Studies of Amyloid Proteins

淀粉样蛋白的固态核磁共振研究

基本信息

批准号：
10687158
负责人：
ROBERT Guy GRIFFIN
金额：
$ 70.85万
依托单位：
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-08-01 至 2027-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10687158
关键词：
2,4-Dinitrophenol Aducanumab Alzheimer&apos s Disease Alzheimer&apos s disease brain Amino Acids Amyloid Amyloid Fibrils Amyloid Proteins Amyloid beta-Protein Amyloidosis Antibodies Arctic mutation Binding Biological Models Brain Chemicals Computer software Coupled Coupling Cryoelectron Microscopy Detection Development Dialysis procedure Early Onset Alzheimer Disease Exhibits Frequencies Genetic Polymorphism Globulins Grant Growth Hydration status Investigation Isotopes Label Magic Measurement Measures Methodology Methods Modeling Molecular Structure Morphology N-terminal NMR Spectroscopy Pathologic Peptides Polymorph Population PrP Probability Process Proteins Publishing Reproducibility Research Resolution Sampling Signal Transduction Spectrum Analysis Structure System Tail Techniques Testing Thermodynamics Torsion Variant amyloid peptide amyloid structure beta-2 Microglobulin catalyst experimental study flexibility in vivo method development mutant next generation paragon solid state nuclear magnetic resonance sup35 tool yeast prion

项目摘要

Project Summary/Abstract The proposed research focuses on the application and development of magic angle spinning (MAS) NMR as a tool for structural investigations of amyloid peptides and proteins. The research covers four major topics. A. Structure of Amyloid Peptides and Proteins (1) Aβ1-42 and Aβ1-40: Using 1H detected and DNP magic angle spinning (MAS) and cryoEM we plan the following experiments on Aβ: (a) a determination of the structure of Aβ1-40; (b) the structure of the pathologically important plaque seeded Aβ1-42; (c) the structure of the N-terminal tail and the binding of antibody Aducanumab to the tail; (d) and the structure of mutants for Aβ1-42. (2) Beta-2-microglobulin (β2m) and DeltaN6-β2m: We plan to determine the structure of the 93 AA β2m-DeltaN6 variant of the dialysis related amyloidosis (DRA) protein. In addition, DeltaN6 is thought to be the catalyst that seeds β2m plaques in-vivo. We therefore also intend to study mixtures of β2m and DeltaN6. (3) Amyloid polymorphism: We intend to determine the structure of the three polymorphs of the amyloidiogenic peptide GNNQQNY from Sup35. These are present in a consistent 1:1:1 population and will provide the first study of the manner in which defined structural changes alter 13C and 15N shifts. 1H, 13C and 17O NMR will be used to characterize the structure around the H2O molecules in the GNNQQNY lattice. B. NMR methods None of the above structural studies would be possible absent NMR methods to assign spectra, measure distances and torsion angles, to enhance signal intensities, etc. We therefore plan to continue the development of the methods essential for these structural investigations. (a) We plan to continue these experiments by initially preparing U-17O/13C/15N GNNQQNY to further develop the spectroscopy and then to apply it to spectroscopy of Aβ1-42 and Aβ1-40. The experiments will employ 1H detection and DNP in order to optimize the sensitivity. PAR and PAIN have been essential to MAS NMR structure determinations but they are semiquantitative methods to measure 13C-13C and 13C-15N distances. Using SPINEVOLUTION software and experiments on model systems, we plan to develop these approaches into quantitative distance measurement techniques.

项目概要/摘要拟研究重点是魔角旋转（MAS）核磁共振的应用和发展作为淀粉样肽和蛋白质结构研究的工具。研究内容涵盖四大主题。 A. 淀粉样肽和蛋白质的结构 (1) Aβ1-42 和 Aβ1-40：使用 1H 检测和 DNP 魔角旋转 (MAS) 和冷冻电镜，我们计划以下关于Aβ的实验：(a)Aβ1-40结构的测定； (b) 结构病理学上重要的斑块接种 Aβ1-42； (c) N-末端尾部的结构和结合尾部抗体 Aducanumab； (d) 和 Aβ1-42 突变体的结构。 (2) Beta-2-微球蛋白(β2m)和DeltaN6-β2m：我们计划确定透析相关淀粉样变性(DRA)蛋白的93AA β2m-DeltaN6变体的结构。此外，DeltaN6 被认为是在体内播种 β2m 斑块的催化剂。因此，我们还打算研究 β2m 和 DeltaN6 的混合物。 (3)淀粉样蛋白多态性：我们打算确定淀粉样蛋白的三种多态性的结构。来自 Sup35 的淀粉样蛋白生成肽 GNNQQNY。它们以一致的 1:1:1 比例存在并将首次研究确定的结构变化改变 13C 和 15N 的方式轮班。 1H、13C 和 17O NMR 将用于表征 H2O 分子周围的结构 GNNQQNY 格子。 B. NMR 方法如果没有 NMR 方法来分配，上述结构研究都是不可能的光谱，测量距离和扭转角，以增强信号强度等。因此我们计划继续开发这些结构研究所必需的方法。（一）我们计划通过初步准备 U-17O/13C/15N GNNQQNY 来继续这些实验，以进一步开发光谱，然后将其应用于 Aβ1-42 和 Aβ1-40 的光谱。实验将采用 1H 检测和 DNP 以优化灵敏度。 PAR 和 PAIN 对 MAS 至关重要 NMR 结构测定，但它们是测量 13C-13C 和 13C-15N 的半定量方法距离。使用 SPINEVOLUTION 软件和模型系统实验，我们计划开发这些方法融入了定量距离测量技术。