Solid State NMR Studies of Amyloid Proteins

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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.