Solid State NMR Studies of Amyloid Proteins

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)Aβ 1-42：我们最近记录了Aβ1-42的MAS光谱，显示出优异的分辨率， 多晶型，并获得了这种有毒形式的初始原子分辨率结构， 阿尔茨海默氏症蛋白质。我们计划：（a）确定Aβ1-42突变体的结构，包括cryoEM （B）确定N-末端的构象及其与Zn 2+的相互作用， 抑制Aβ1-42原纤维增殖的伴侣蛋白Brichos，和（c）人AD脑的种子原纤维 提取物，以确定Aβ1-42的病理重要结构，并确定其结构 (2)β 2-微球蛋白（β 2 m）和ΔN6-β 2 m：99 AA和93 AA ΔN6变体的β 2-微球蛋白（β 2 m）和ΔN6-β 2 m： 透析相关的淀粉样变性（淀粉样变性）蛋白质大部分是完整的。此外，我们确定， 股是PIR，并已获得足够的约束，以确定初步结构。我们计划 以完成具有附加距离和扭转角测量的结构。 (3)淀粉样蛋白中的水层：我们打算确定TTR 105 -115纤维中的水双层结构 在GNNQQNY晶体中，这似乎是淀粉样纤维和“胶”的共同特征， 结合原纤维形成原纤维。在这些研究中将使用1H、2 H和17 O MAS实验。一部分 在这项工作中，我们计划开发新的仪器和脉冲序列来观测17 O 最终产生5D DNP增强的e-/1H/17 O/13 C/15 N相关光谱。 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。PAR和 PAIN对于MAS NMR结构测定是必不可少的，但它们是半定量的 测量13 C-13 C和13 C-15 N距离的方法。使用SPINEVOLUTION和SIMPSON 软件和模型系统实验，我们计划将这些方法发展为定量的 距离测量技术。1H检测将集成到这些第二阶偶极中 用于最佳有效结构研究的再耦合技术。

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) Aβ 1-42: We have recently recorded MAS spectra of Aβ1-42 that exhibit excellent resolution, a single polymorph, and have obtained the initial atomic resolution structure of this toxic form of this Alzheimer's protein. We plan to: (a) determine the structure of mutants for Aβ1-42 including a cryoEM studies of the fibrils; (b) determine the conformation of the N-terminus and its interaction with Zn2+ the chaperone Brichos that inhibits propagation of Aβ1-42 fibrils, and (c) seed fibrils with human AD brain extracts to determine the pathologically important structure of Aβ1-42 and determine their structures (2) Beta-2-microglobulin (β2m) and ΔN6-β2m: Assignments for these 99 AA and 93 AA ΔN6 variant dialysis related amyloidosis (DRA) proteins are largely complete. In addition, we established that the strands are PIR, and have obtained sufficient constraints to determine a preliminary structure. We plan to complete the structure with additional distances and torsion angle measurements. (3) Water layer in amyloid: We intend to determine the structure of the water bilayer in TTR105-115 fibrils and in GNNQQNY crystals, which appear to be a common feature of amyloid fibrils and the “glue” that binds protofibrils to form fibrils. 1H, 2H and 17O MAS experiments will be used in these studies. As part of this effort we plan to develop new instrumentation and pulse sequences for observation of 17O ultimately yielding 5D DNP enhanced e-/1H/17O/13C/15N correlation spectra. 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. 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 and SIMPSON software and experiments on model systems, we plan to develop these approaches into quantitative distance measurement techniques. 1H detection will be integrated into these 2nd order dipolar recoupling techniques for optimally efficient structural studies.