Tuesday, May 7, 2024
5/7/2024
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) Aß 1-42: We have recently recorded MAS spectra of Aß1-42 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 Aß1-42 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 Aß1-42 fibrils, and (c) seed fibrils with human AD brain extracts to determine the pathologically important structure of Aß1-42 and determine their structures (2) Beta-2-microglobulin (ß2m) and ¿N6-ß2m: Assignments for these 99 AA and 93 AA ¿N6 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.
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