Biomolecular NMR spectroscopy has limitations in the determination of protein structures: an inherent size limit and the requirement for expensive and potentially difficult isotope labelling pose considerable hurdles. Therefore, structural analysis of larger proteins is almost exclusively performed by crystallography. However, the diversity of biological NMR applications outperforms that of any other structural biology technique. For the characterization of transient complexes formed by proteins and small ligands, notably oligosaccharides, one NMR technique has recently proven to be particularly powerful: saturation-transfer difference NMR (STD-NMR) spectroscopy. STD-NMR experiments are fast and simple to set up, with no general protein size limit and no requirement for isotope labelling. The method performs best in the moderate-to-low affinity range that is of interest in most of glycobiology. With small amounts of unlabelled protein, STD-NMR experiments can identify hits from mixtures of potential ligands, characterize mutant proteins and pinpoint binding epitopes on the ligand side. STD-NMR can thus be employed to complement and improve protein–ligand complex models obtained by other structural biology techniques or by purely computational means. With a set of protein–glycan interactions from our own work, this review provides an introduction to the technique for structural biologists. It exemplifies how crystallography and STD-NMR can be combined to elucidate protein–glycan (and other protein–ligand) interactions in atomic detail, and how the technique can extend structural biology from simplified systems amenable to crystallization to more complex biological entities such as membranes, live viruses or entire cells.
Ligand Screening by Saturation-Transfer Difference (STD) NMR Spectroscopy
