R01AG058504

Solid state NMR studies of amyloid proteins - 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) A1-42 and A1-40: Using 1H detected and DNP magic angle spinning (MAS) and cryoEM, we plan the following experiments on A:
(A) Determination of the structure of A1-40
(B) The structure of the pathologically important plaque seeded A1-42
(C) The structure of the N-terminal tail and the binding of antibody aducanumab to the tail
(D) The structure of mutants for A1-42

(2) Beta-2-microglobulin (2M) and N6-2M: We plan to determine the structure of the 93 AA 2M-N6 variant of the dialysis related amyloidosis (DRA) protein. In addition, N6 is thought to be the catalyst that seeds 2M plaques in-vivo. We therefore also intend to study mixtures of 2M and N6.

(3) Amyloid polymorphism: We intend to determine the structure of the three polymorphs of the amyloidogenic 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 A1-42 and A1-40. 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.

Massachusetts Institute Of Technology

NOT APPLICABLE

93.866 - Aging Research

National Institute on Aging (NIA) [HHS - NIH]

Cambridge, Massachusetts 021394301 United States

Single Zip Code

NIH Research Project Grant (Parent R01 Clinical Trial Not Allowed) (PA-20-185)

Amendment Since initial award the total obligations have increased 396% from $709,495 to $3,522,439.