Direct Observation of Carbohydrate Hydroxyl Protons in Hydrogen Bonds with a Protein

Hydroxyl proton resonances of uniformly ¹³C-labeled Manα(1-2)Manα(1-2)ManαOMe (Man₃) bound to cyanovirin-N (CV-N) were detected at ambient temperature in aqueous solution by NMR spectroscopy. The directions of the hydroxyl groups were determined on the basis of NOEs, and a previously unknown hydrogen-bonding network between Man₃ and CV-N was discovered. This is the first report on detecting hydroxyl protons of a protein-bound carbohydrate in aqueous solution by NMR. Approaches such as those presented here may open the door for accurately determining intermolecular hydrogen bonds in carbohydrate-protein complexes.