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.

项目摘要/摘要拟议的研究重点是魔术角旋转（MAS）NMR的应用和开发作为淀粉样蛋白质和蛋白质结构投资的工具。该研究涵盖了四个主要主题。 A.淀粉样肽和蛋白质的结构（1）Aβ1-42和Aβ1-40：使用1H检测到DNP魔法旋转（MAS）和冷冻，我们计划经过Aβ的实验：（a）测定Aβ1-40的结构；（b）结构病理上重要的斑块种子Aβ1-42；（c）N端尾的结构和结合尾巴的抗体aducanumab；（d）和Aβ1-42突变体的结构。（2）β-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93AAβ2M-deltAn6变体的结构。此外，deltan6被认为是催化剂在体体内播种β2M斑块。因此，我们还打算研究β2M和Deltan6的混合物。（3）淀粉样多态性：我们打算确定三个多晶型物的结构 SUP35的淀粉样蛋白生成肽GNNQQNY。这些存在于一致的1：1：1人群中并将提供对定义结构变化改变13C和15N的方式的首次研究转移。 1H，13C和17O NMR将用于表征H2O分子周围的结构 Gnnqqny晶格。 B. NMR方法没有上述结构研究，没有NMR方法可以分配光谱，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投资必不可少的方法。（a）我们计划首先准备U-17O/13C/15N GNNQQNY以进一步发展这些实验光谱法然后将其应用于Aβ1-42和Aβ1-40的光谱法。实验将员工1H 检测和DNP以优化灵敏度。标准杆和痛苦对于MAS至关重要 NMR结构确定，但它们是测量13C-13C和13C-15N的半定量方法距离。我们使用模型系统上的Spinevolution软件和实验，我们计划开发这些方法是定量距离测量技术。