Solid State NMR Studies of Amyloid Proteins

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) Aβ 1-42：我们最近记录了 Aβ1-42 的 MAS 谱，该谱表现出优异的分辨率，单 多晶型物，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，包括冷冻电镜 原纤维的研究； (b) 确定 N 端的构象及其与 Zn2 的相互作用 伴侣 Brichos 抑制 Aβ1-42 原纤维的增殖，以及 (c) 人类 AD 大脑中的种子原纤维 提取物确定 Aβ1-42 的病理重要结构并确定其结构 (2) Beta-2-微球蛋白 (β2m) 和 ΔN6-β2m：这些 99 AA 和 93 AA ΔN6 变体的分配 透析相关淀粉样变性 (DRA) 蛋白基本完整。此外，我们确定 链是PIR，并且已经获得足够的约束来确定初步结构。我们计划 通过额外的距离和扭转角测量来完成结构。 (3)淀粉样蛋白中的水层：我们打算确定TTR105-115原纤维中水双层的结构 在 GNNQQNY 晶体中，这似乎是淀粉样原纤维的共同特征，也是淀粉样蛋白原纤维的“粘合剂” 结合原纤维形成原纤维。这些研究中将使用 1H、2H 和 17O MAS 实验。作为一部分 在这项工作中，我们计划开发新的仪器和脉冲序列来观测 17O 最终产生 5D DNP 增强的 e-/1H/17O/13C/15N 相关光谱。 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。 PAR 和 PAIN 对于 MAS NMR 结构测定至关重要，但它们是半定量的 测量 13C-13C 和 13C-15N 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开发成定量方法 距离测量技术。 1H 检测将集成到这些二阶偶极子中 用于最佳有效结构研究的重新耦合技术。

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.