Solid State NMR Studies of Amyloid Proteins

提出的研究重点是魔角旋转核磁共振技术的应用和发展。 作为研究淀粉样多肽和蛋白质结构的工具。这项研究涵盖了四个主要方面 主题。 A.淀粉样多肽和蛋白质的结构 (1)Aβ1-42：我们最近记录了Aβ1-42的主吸收光谱，显示出极好的分辨率，单个 多晶型，并获得了这种有毒形式的初步原子分辨结构 老年痴呆症蛋白质。我们计划：(A)确定Aβ1-42突变体的结构，包括低温EM 纤维的研究；(B)测定N-末端的构象及其与锌的相互作用 伴侣Brichos抑制Aβ1-42纤维的繁殖，以及(C)人类AD脑的种子纤维 确定Aβ1-42重要病理结构并确定其结构的提取物 (2)β-2-微球蛋白(β2M)和ΔN6-β2M：99个氨基酸和93个氨基酸的ΔN6变异体的归属 透析相关的淀粉样变性(DRA)蛋白大部分是完整的。此外，我们还确定， 链是PIR，并且已经获得了足够的限制来确定初步结构。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纤维中的水双层结构 在GNNQQNY晶体中，这似乎是淀粉样纤维的共同特征，以及 结合原纤维形成纤维。在这些研究中将使用1H、2H和17O MAS实验。作为一部分 在这项工作中，我们计划开发用于观测17O的新仪器和脉冲序列 最终得到5DDNP增强的e/1H/17O/13C/15N相关谱。 B.核磁共振方法如果没有核磁共振方法，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 继续发展对这些结构调查至关重要的方法。标准杆和 疼痛对于MAS核磁共振结构测定是必不可少的，但它们是半定量的 方法测量13C-13C和13C-15N距离。使用旋转法和辛普森法 软件和模型系统上的实验，我们计划将这些方法发展成定量的 距离测量技术。1H检测将集成到这些2阶偶极子中 用于最优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.