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Abstract
Novel nuclear magnetic resonance (NMR) methods have been developed for structure and function studies of sialyltransferases interacting with synthetic substrates. The presented methodology has been applied to a mammalian sialyltransferase ST6Gal-1 which catalyzes the transfer of sialic acids to the terminal galactose of carbohydrates on the cell surface. Conventional structural methods have failed to produce a structure of ST6Gal-1 because of its membrane association, native glycosylation, and poor expression in bacterial hosts. A new methodology is urgently needed to provide a precise molecular structure of ST6Gal-1 and the geometries of bound ligands. The provided information can be used in the design of specific inhibitors to regulate the distribution of sialic acids in biological systems. NMR is a unique tool to study a broad range of proteins that resist study by X-ray crystallography. The methodology takes advantage of isotopically-labeled and nitroxide-labeled substrates which can probe geometric properties of the active site of a protein. Protein-ligand interactions are usually the key to understanding the function of enzymes. This work investigated the conformation, orientation, kinetics, and protein-contact surfaces of bound ligands. When nitroxide-labeled substrates and isotopically-labeled protein were utilized, five phenylalanines in and near the protein active site were identified and their distances to the substrates were calculated based on NMR data. This distance-sensitive nitroxide-labeled probe also was used to derive the relative placement of substrates simultaneously present in the active site of ST6Gal-1. The above approach has improved our structural understanding of ST6Gal-1 and provided an important step toward the structure-based design of efficient inhibitors. The synthetic strategies and synthesized compounds are applicable to other sialyltransferases, and the presented NMR labeling methods will be highly valuable for studying the structures of various glycoproteins.