NEW SOLID STATE NMR METHODOLOGY FOR STRUCTURAL STUDIES OF BIOPOLYMERS

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In the past year, we have made substantial progress in the following two areas:1. Development of Multiple Quantum Carbon-13 Solid State NMR and Application to beta-Amyloid Fibril Structure We have developed solid state NMR techniques that allow the detection of multiple quantum NMR signals in carbon-13-labeled biopolymer samples. These multiple quantum signals arise from collective transitions of groups of dipole- coupled carbon-13 nuclear spins. Observation of multiple quantum signals requires that the carbon-13 nuclei be within approximately 5 angstroms of one another. Therefore, multiple quantum spectra can be used as a probe of biopolymer conformation or intermolecular packing in noncrystalline solids. We have applied the new multiple quantum NMR techniques in structural studies of fibrils formed by the 40-residue beta-amyloid peptide of Alzheimers diseases, as well as fibrils of shorter model peptides derived from the full-length beta-amyloid peptide. Comparison of experimental multiple quantum signal intensities with numerical simulations indicates unambiguously that the beta- amyloid peptides are arranged in a parallel beta-sheet structure in the fibrils. These results rule out existing structural models for beta- amyloid fibrils that are based on an anti-parallel arrangement. 2. Development of Multidimensional Solid State NMR Techniques for Structural Studies of Uniformly Labeled, Oriented Peptides and Proteins We have developed two- and three-dimensional NMR spectroscopic techniques that allow the resolution and sequential assignment of NMR signals in uniformly carbon-13 and nitrogen-15 labeled peptides and proteins that are uniaxially oriented with respect to the magnetic field of the solid state NMR spectrometer. These techniques overcome major hurdles to the determination of full structures of peptides and proteins, including membrane-bound peptides and proteins, by solid state NMR. The new techniques depend on novel homonuclear and heteronuclear decoupling sequences that permit the observation of carbon-13 and nitrogen-15 chemical shifts in the presence of strong dipole-dipole couplings that would otherwise render the spectra intractable. The new techniques have been demonstrated on simple model peptides in single crystal form. Extensions to bona fide proteins are in progress. - nuclear magnetic resonance, protein structure