DETERMINATION OF 3D-DIMENSIONAL STRUCTURES OF MACROMOLECULES IN SOLUTION BY NMR

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Work in this laboratory has been focussed on the determination of three-dimensional structures of larger proteins in solution by NMR, with a particular emphasis on cytokines, immune related proteins, DNA-protein complexes, and protein-protein complexes. A considerable effort has been placed on the development of three- and four-dimensional heteronuclear NMR to extend the application of NMR as a method for determining three-dimensional structures of proteins in solution beyond the limits of conventional two-dimensional NMR (about 100 residues) to molecules in the 150- to 400-residue range. High resolution solution structures of a number proteins have been determined. These include the oligomerization domain of p53, the beta chemokine macrophage inflammatory protein-1-beta (hMIP-1-beta), oxidized and reduced human thioredoxin, and the trypsin inhibitor of Ascaris. Extensive use in these studies has been made of multi-dimensional heteronuclear NMR and of systematic conformational searches to obtain stereospecific assignments and torsion angle restraints which have enabled us to obtain very high resolution structures comparable in accuracy to 2 Angstroms resolution X-ray structures. The typical accuracy attainable is 0.2-0.4 Angstroms for the backbone atoms and 0.3-0.5 Angstroms for the internal side chains.