C-13 NMR-STUDIES OF WHEAT-GERM-AGGLUTININ INTERACTIONS WITH N-ACETYLGLUCOSAMINE AT A MAGNETICALLY ORIENTED BILAYER SURFACE

The orientation of synthetic C-13-labeled glycolipid receptors and the ir interaction with the plant lectin wheat germ agglutinin have been s tudied in an oriented membrane system using NMR spectroscopy. A series of -C-13(2)]acetamido-2-deoxy-beta-D-glucopyranosides were synthesize d with between zero and four hydrophilic ethoxy units between the head group and an alkyl chain which anchors the receptors in the bilayers. The chemical shift anisotropy of the C-13 carbonyl and a C-13-C-13 dip olar coupling between the labeled carbons provide information about th e orientation and dynamics of the receptor headgroup in oriented membr ane systems. It was found that the headgroups of the receptors with tw o, three, or four ethoxy units appeared isotropic when incorporated in the oriented bilayers, but those of the receptors with zero or one et hoxy units were significantly ordered by the bilayers. The average ori entations consistent with measured spectral parameters were determined for the receptors with zero and one ethoxy units and were found to co incide with low-energy conformations from molecular modeling. When the plant lectin wheat germ agglutinin was added to the sample, only the receptors with two, three, or four ethoxy units separating the headgro up from the alkyl chain showed evidence of binding by the lectin. Alth ough the C-13-labeled resonances broadened when the protein bound, no changes in dipolar couplings or chemical shift anisotropies could be d etected, suggesting that the motion of the headgroup was slowed by pro tein binding, but average orientation and overall order changed little . Competition studies demonstrated that none of the lectin/receptor co mplexes are more stable than the complex of the lectin and N-acetylglu cosamine in solution. These results suggest that the membrane does not stabilize the interactions of wheat germ agglutinin with these cell-s urface receptors. Furthermore, molecular modeling demonstrates that th e zero- and one-spacer receptors may not bind wheat germ agglutinin be cause the orientations of the N-acetyl groups in these receptors would result in significant steric contacts between the lectin/receptor com plex and the membrane.