AN INVESTIGATION OF THE LIGAND-BINDING SITE OF THE GLUTAMINE-BINDING PROTEIN OF ESCHERICHIA-COLI USING ROTATIONAL-ECHO DOUBLE-RESONANCE NMR

Glutamine-binding protein (GlnBP) is an essential component of the glu tamine transport system in Escherichia coli. Rotational-echo double-re sonance (REDOR) solid-state nuclear magnetic resonance (NMR) has been used to determine internuclear distances in the complex of GlnBP and i ts ligand, L-glutamine. REDOR, combined with strategically placed isot opic labels, is effective in obtaining model-independent internuclear distances and thus detailed structural information on the ligand-bindi ng site of GlnBP. The existence of a single histidine residue (His156) in the binding site has provided an excellent probe for distance meas urements between protein and ligand. REDOR distances up to 6.3 Angstro m have been observed between C-13 labels in L-glutamine and N-15 label s in His156. These results have unambiguously determined the ligand or ientation with respect to the imidazole ring of His156, which is an im portant first step in refining the ligand-binding-site model of GlnBP in general. The measured distances were also used as constraints in re strained molecular dynamics calculations of the complex using the unli ganded crystal structure of GlnBP as the starting point. The simulatio ns clearly show consistency between calculated distances and those mea sured by REDOR.