NUCLEAR-MAGNETIC-RESONANCE STRUCTURAL AND LIGAND-BINDING STUDIES OF BLBC, A 2-DOMAIN FRAGMENT OF BARLEY LECTIN

Plant lectins are useful targets for biophysical studies of protein-ca rbohydrate recognition, a process of general interest because of its m any roles in human physiology. Here, nuclear magnetic resonance (NMR) based structural and carbohydrate binding data on a two-domain fragmen t of the normally four-domain barley lectin protein are presented. The structural data, while preliminary, clearly shows that the recombinan tly produced simplified model system, called BLBC, retains a nativelik e fold, However, unlike the full-length parent protein, which is dimer ic, BLBC is shown by pulsed-field gradient NMR diffusion studies to be largely monomeric. Still, the fragment retains nativelike carbohydrat e binding properties, These properties are examined in some detail usi ng heteronuclear single quantum coherence (HSQC) NMR spectroscopy on a uniformly N-15-labeled sample. Ligand-induced chemical shift changes in the H-1-N-15 HSQC spectrum are monitored as N-15-labeled BLBC is ti trated with increasing concentrations of the unlabeled carbohydrate, N ,N',N''-triacetylchitotriose. Well-resolved resonances from the indivi dual domains show that BLBC binds ligand at two distinct and independe nt ligand binding sites, one in each domain, Binding constants of (1.1 +/- 0.2) x 10(3) M-1 and (0.6 +/- 0.2) x 10(3) M-1 are determined for the B and C domain sites, respectively, These results are discussed i n relation to ligand binding studies that have previously been carried out on a highly homologous protein, wheat germ agglutinin.