Progress in FY 2003 has been in the following areas: 1. Methodology: We have developed new solid state NMR techniques for measuring interatomic distances in peptide and protein samples that are prepared with uniformly 13C-enriched amino acids. These techniques include a new version of """"""""rotational resonance"""""""", in which radio-frequency pulse sequences are applied to restore nuclear magnetic dipole-dipole couplings under magic-angle spinning conditions, with the """"""""rotational resonance"""""""" bandwidth being controlled by the details of the pulse sequences. We have also developed a new form of two-dimensional 13C-13C NMR spectroscopy in which 13C-13C chemical shift correlations are mediated by proton nuclei. This technique allows long-range 13C-13C contacts to be detected in solid state NMR spectra. In addition, we have developed experimental techniques for extracting structural constraints from NMR spectra of fibrillar peptide samples deposited on planar substrates, and mathematical methods for analyzing such spectra. 2. Amyloid fibril structure: We have developed experimental conditions for preparation of amyloid fibrils from the 40-residue beta-amyloid peptide associated with Alzheimer's disease that permit control of fibril morphology and molecular structure. Depending on conditions, two distinct fibril morphologies can be prepared, which differ in their appearance in electron microscopy and in their NMR signals. Structural differences are heritable, in that """"""""seeds"""""""" from a given preparation can be used to propagate the morphology and molecular structure. This finding has significance for understanding of the phenomenon of """"""""strains"""""""" in prion diseases. Preliminary tests of fibril toxicity in rat hippocampal cell cultures indicate that different fibril morphologies have different toxicities. We have also completed a careful study of the beta-sheet structure in fibrils formed by residues 11-25 of the full-length beta-amyloid peptide, showing that the registry of hydrogen bonding in the antiparallel beta-sheets is well-ordered, but pH dependent. 3. Studies of an HIV-1 related peptide/antibody complex: We have completed investigations of the bound conformation of a peptide derived from the V3 loop of HIV-1 gp120 in complexes with a broadly neutralizing human monoclonal anti-gp120 antibody (Fv fragements). In this project, the peptide is uniformly 13C- and 15N-labeled at seven residues in the epitope region, allowing conformational measurements by our newly-developed methods appropriate for samples with uniformly labeled residues. Our measurements indicate a hairpin structure for the bound peptide. These are the first solid state NMR studies of a peptide/antibody complex with uniformly labeled peptides, setting the stage for future studies of complexes between peptides and integral membrane receptor proteins. 4. Studies of protein folding by solid state NMR: We have carried out initial studies of the conformational distibutions of a model protein (villin HP35) in frozen solution under denaturing conditions. These studies show how solid state NMR can characterize protein folding and unfolding at a site-specific level. Preliminary results indicate that the three helices in HP35 unfold at different points and to different extents with increasing denaturant concentration, calling into question common two-state models of folding.
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