MULTIDIMENSIONAL H-1 AND N-15 NMR INVESTIGATION OF GLUTAMINE-BINDING PROTEIN OF ESCHERICHIA-COLI

Specific and uniform N-15 labelings along with site-directed mutagenes is of glutamine-binding protein have been utilized to obtain assignmen ts of the His156, Trp32 and Trp220 residues. These assignments have be en made not only to further study the importance of these 3 amino acid residues in protein-ligand and protein-protein interactions associate d with the active transport of L-glutamine across the cytoplasmic memb rane of Escherichia coli, but also to serve as the starting points in the sequence-specific backbone assignment. The assignment of H(epsilon -2) of His156 refines the earlier model where this particular proton f orms an intermolecular hydrogen bond to the delta-carbonyl of L-glutam ine, while assignments of both Trp32 and Trp220 show the variation in local structures which ensure the specificity in ligand binding and pr otein-protein interaction. Using 3D NOESY-HMQC NMR, amide connectiviti es can be traced along 8-9 amino acid residues at a time. This paper i llustrates the usefulness of combining N-15 isotopic labeling and mult inuclear, multidimensional NMR techniques for a structural investigati on of a protein with a molecular weight of 25 000.