The proposed research focuses on the application and development of magic angle spinning (MAS) NMR as a tool for structural investigations of peptides and proteins. The research covers two broad areas. A. Structure of Amyloid Peptides and Proteins (1) Water layer in TTR105-115 : We intend to determine the structure of a water bilayer in TTR105-115 fibers, which may be a common feature of amyloid fibrils and the """"""""glue"""""""" that binds protofibrils to form fibrils. (2) PI3-SH3: We completed spectral assignments of the fibrillar form of this 86 AA protein, and established that the strands are in a parallel in-registe (PIR) arrangement with DNP. To complete the structure we need to measure additional interstrand contacts and sidechain distances and angles. (3) Beta-2-microglobulin (?2m): Assignments for this 99 AA protein are complete and we established that the strands are PIR. We have detected some interstrand contacts. To complete the structure we require additional distances and torsion angles. We also intend to study the ?N6 construct of ?2m. (4) CsgA and CsgB - functional amyloids: Recently it has become clear that some amyloids are functional, as opposed to pathological. We plan to study one of these, CagA, that is associated with biofilms and thought to form a ?-helical structure. 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. (1) Recoupling in 2H labeled proteins DNP experiments function optimally with 2H labeled proteins. We there plan to develop methods for 2H-13C/15N recoupling based on third spin assisted experiments. (2) Spin diffusion and PAR and PAIN Simulations is used extensively in ssNMR as a semiquantitative method to measure distances. Using SPINEVOLUTION software we plan to improve the accuracy of distance measurements and apply the results to proteins.
The research proposes to determine high resolution molecular structures of amyloid peptides and proteins associated with phosphatidyl inositol-3-kinase SH3 domain, dialysis related amyloidosis (beta-2 microglobulin) CsgA as well as some model amyloid systems. In addition we propose to continue to develop the methodology to perform the structural experiments focusing on 2H recoupling and simulations of spin diffusion.
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