This project is directed at examining both primary structures and three-dimensional (3D) structures of macromolecules in their active states. The Laboratory utilizes established NMR techniques and develops new techniques to accomplish this goal. New developments in the past year include implementation of three-dimensional NMR experiments and isotopic labelling methods, which have been developed in this lab for polysaccharides and, using existing methods for protein labelling. Computational methods include 3D NMR data processing, theoretical analysis of multispin NOE behavior, distance geometry structure refinement, and molecular modeling. Our efforts in DNA oligonucleotides have focused on using NMR parameters, interatomic distances and coupling constants, in the determination of solution-state 3D structures. These studies have been directed at several oligonucleotide-Daunomycin complexes. It has been observed that drug binding is sequence specific and that the small oligonucleotide (dCGTACG)2-Daunomycin complex, for which there exists a crystal structure, exhibits exchange broadening in solution. This problem is circumvented by longer oligonucleotide sequences and three dimensional structural characterization is underway. Theoretical comparisons of NOE buildup behavior between the crystal structure and regular DNA structures indicate that they are readily distinguished by NMR spectroscopy. DNA-protein interaction studies are underway on a small DNA-binding peptide as well as two small DNA-binding proteins which are being cloned for large scale production, istopic, labelling and NMR structural analyses. The determination of oligo- and polysaccharide primary structures is important for categorization and understanding biological function, e.g. cross-reactivity. We have extended previous work on indirect-detected 1H NMR experiments to include 3D-NMR and 13C isotopic labelling techniques. The 3D spectra of labelled carbohydrates shows great potential for overcoming problems in this type of analysis. Work is continuing on several poly- and oligosaccharide structures using the 3D methods. This approach also shows potential for examining glycoprotein systems and extensions to include computational methods to examine 3D structures to gain further insight into cross-reactivity and the antigenic response.
