HYDROGEN-BONDING GEOMETRY OF A PROTEIN-BOUND CARBOHYDRATE FROM WATER EXCHANGE-MEDIATED CROSS-RELAXATION

We present heteronuclear two-dimensional methods for the analysis of t he geometry of exchangeable protons on a protein-bound carbohydrate. B y using a water-selective NOESY-HSQC, we observed cross-relaxation bet ween carbohydrate hydroxyl protons and non-exchangeable ring protons i n the complex of [C-13(6)]-alpha-methyl-D-mannopyranoside with recombi nant rat mannose binding protein. Using a simple kinetic model, we wer e able to explain the differences in the initial slopes of the resulti ng cross-relaxation buildup curves in terms of the geometry of the hyd roxyl protons in the bound state. The hydroxyl rotamers consistent wit h our cross-relaxation data fit very well with predictions based on th e crystal structure of MBP bound to a mannose-rich oligosaccharide. Th ese methods should be applicable to other systems where both ligand ex change and water exchange are fast relative to the rate of cross-relax ation.