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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.

项目总结/摘要本文主要研究魔角旋转核磁共振技术的应用和发展作为淀粉样肽和蛋白质结构研究的工具。该研究涵盖了四个主要领域。话题 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cryoEM，我们计划对Aβ进行以下实验：（a）Aβ1-40的结构测定;（B）A β1-40的结构测定。病理上重要的空斑接种Aβ1-42;（c）N-末端尾部的结构和抗体Aducanumab的尾部;（d）Aβ1-42突变体的结构。 (2)β-2-微球蛋白（β 2 m）和DeltaN 6-β 2 m：我们计划确定透析相关淀粉样变性（AAP）蛋白的93 AA β 2 m-DeltaN 6变体的结构。此外，DeltaN 6被认为是体内接种β 2 m斑块的催化剂。因此，我们还打算研究β 2 m和DeltaN 6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晶型物的结构。来自Sup 35的淀粉样蛋白生成肽GNNQQNY。这些都存在于一个一致的1：1：1人口并将首次研究确定的结构变化改变13 C和15 N的方式运动一样的1H、13 C和17 O NMR将用于表征H2O分子周围的结构， GNNQQNY晶格。 B。如果没有NMR方法，上述结构研究都不可能进行。光谱，测量距离和扭转角，以增强信号强度等。因此，我们计划继续发展这些结构调查所必需的方法。(a)我们计划继续这些实验，首先制备铀-17 O/13 C/15 N GNNQQNY，并将其应用于Aβ1-42和Aβ1-40的光谱分析。实验将使用1H 检测和DNP，以优化灵敏度。PAR和Pain对于MAS至关重要 NMR结构测定，但它们是测量13 C-13 C和13 C-15 N的半定量方法的距离.使用SPINEVOLUTION软件和模型系统的实验，我们计划开发这些方法转化为定量距离测量技术。