Nuclear Magnetic Resonance (NMR) can provide invaluable data on the position and conformation of ligands bound to proteins.  The conformational data often come from transferred NOEs (trNOEs) and saturation transfer difference (STD) spectra.  Positions on the protein surface often come from chemical shift perturbation of assigned protein resonances and intermolecular NOEs.  However, when ligands are glycans and proteins are large or themselves glycosylated, data from these traditional methods are often limited.  Full assignment of protein resonances is often difficult, particularly for glycosylated proteins which are best expressed in mammalian cells, and intermolecular NOEs are often sparse due to the diminished contribution of hydrophobic terms to interaction energies. <br/>            We have developed a procedure that combines a new strategy for the assignment of resonances from proteins sparsely labeled with single or multiple types of isotope-enriched amino acids, with perturbation data from paramagnetic tags attached to either the ligand or the protein, to determine bound-ligand geometry.  The isotopic labeling procedure is readily applicable to glycosylated proteins expressed in mammalian cells, and perturbations of ligand resonances from the paramagnetic tags provide long-range constraints on the ligand position on the protein surface.  We will illustrate this procedure with applications drawn from proteins that bind glycosaminoglycans (fragments from heparin sulfate or chondroitin sulfate) and enzymes that produce complex glycans with extraordinary specificity.<br/>

Bound geometry of glycans using proteins with paramagnetic tags

254th National Meeting (2017)

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