Solid State NMR Studies of Peptides and Proteins

Massachusetts Institute Of Technology, Cambridge MA

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Abstract

[unreadable] DESCRIPTION (provided by applicant): The proposed research focuses on the continued application and development of solid state NMR (ssNMR) as a tool for structural investigations of peptides and proteins. The research covers two broad areas. [unreadable] [unreadable] Structure of Peptides and Proteins [unreadable] [unreadable] (1) With ssNMR measurements of intermolecular distances on both the WT TTR and L111M mutant peptides from transthyretin we plan to refine the structures of the amyloid protofilaments formed by these two peptides. These structures represent the initial high resolution structure of amyloid fibrils. [unreadable] (2) We plan to determine the molecular structure of the monoclinic and orthorhombic crystals formed by the prion peptide GNNQQNY and its three structurally distinct amyloid fibrils. [unreadable] (3) The protein PI3-SH3 forms well-ordered amyloid fibrils that can be uniformly or sparsely 13C,15N labeled. Our goal is to determine the high-resolution structure of the PI3-SH3 amyloid fibril using dipole recoupling. High field EPR will be used to measure distances between monomers. [unreadable] (4) beta-2-microglobulin ([unreadable]2m) is associated with dialysis related amyloidosis (DRA). We plan to determine the structure of the fibrillized form of this protein and compare it to its native structure. [unreadable] (5) We plan to determine a high resolution structures of at least one peptide derived from A[unreadable] (LMVGGVVIA) and A[unreadable] itself using mutants which yield spectra with improved resolution. [unreadable] (6) Two nanocrystalline proteins, GB1 and Crh, are ideal systems to validate new NMR methods and provide a platform for establishing robust and efficient methodology that can be utilized for structure determination of amyloid proteins. We plan to utilize these two systems in this manner. [unreadable] [unreadable] NMR Methods for Protein Structure Determination [unreadable] [unreadable] (1) Recently we showed that two techniques (CMAR and RFDR) perform 13C-13C recoupling and 1H-13C decoupling simultaneously. We plan to extend the development of the new methods and use them to measure distances for structural studies by optimizing the shape of the CMAR sweep for distance measurements. PAIN-CP 13C-15N recoupling experiments will be developed for experiments at high field and high spinning frequency. [unreadable] (2) Spin diffusion 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. [unreadable] (3) 17O NMR experiments offer the possibility of a significant increase in resolution at high field. We plan to develop methods to perform 17O-13C and 17O-15N correlation experiments that will add another important spectral dimension to protein NMR experiments. [unreadable] [unreadable] [unreadable]