The proposed research focuses on the continued application and development of solid state NMR as a tool for structural investigations of peptides and proteins. The research is focused in two different areas. (1) Amyloid Peptides and Proteins and Nanocrystalline Proteins: During the past grant period we completed the first structure determination of a tripeptide, f-MLF-OH, and the monomer of the 11-mer peptide, YTIAALLSPYS (TTR105-115), in the form of an amyloid fibril using dipolar recoupling. We plan to extend these investigations to other systems including: (a) GFAD a small peptide with a known crystal structure that is useful for developing methods to measure interstrand and intersheet distances, (b) a peptide from the Sup35 prion protein (GNNQQNY) crucial in initiating amyloid formation, (c) the L111M mutant of TTR105-115, (d) amylin, (e) a coiled coil peptide that transforms to amyloid (CCbetaPep), and (f) to an amyloidiogenic SH3 domain from phosphatidyl inositol 3-kinase (PI3-SH3). In all of these experiments we will be focusing on intra- and intermolecular structures. In addition, we plan investigations of alpha-spectrin SH3 (alphaSSH3) that will lead to a high resolution structure of this protein. These experiments include the application of existing methods for measuring 13C-15N and 13C-13C distances and torsion angles that have not been tested or applied to larger systems. Further, we plan to use this system to develop cryoprotectants for low temperature spectroscopy. (2) Solid State NMR Methodology: We plan to continue to improve and to develop new approaches for structural studies of peptides and proteins with experiments in five different areas: (a) 13C-13C distance measurements in uniformly labeled systems with a particular focus on 13CH3-13CH3 distances; (b) generalized torsion angle experiments to determine angles in cases that are not accessible with the available techniques; (c) We plan to correlate the orientation of the shift and dipolar tensors to determine the orientation of Cgamma aromatic ring tensor relative to the -CbetaH2- dipolar tensor and therefore to constrain khi2; (d) We plan to develop methods to measure 17O shifts at high field and 17O-13C and 17O-15N distances in peptides and proteins. These will involve REDOR type experiments on labeled peptides, and will utilize dynamic nuclear polarization (DNP) to enhance the signal intensities; (e) With via magnetic dilution and high frequency MAS, we observe 1H spectra of solids that exhibit excellent resolution increased signal-to-noise via 1H detection. We plan to perform additional experiments with magnetic dilution of 1H and indirect detection at 900 MHz and high spinning frequencies.
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