A new combined computational and NMR-spectroscopical strategy for the identification of additional conformational constraints of the bound ligand in an aprotic solvent

This study documents the feasibility of switching to an aprotic medium in s ugar receptor research. The solvent change offers additional insights into mechanistic details of receptor-carbohydrate ligand interactions. If a rece ptor retained binding capacity in an aprotic medium, solvent-exchangeable p rotons of the ligand would not undergo transfer and could act as additional sensors, thus improving the level of reliability in conformational analysi s. To probe this possibility, we first focused on hevein, the smallest lect in found in nature. The NMR-spectroscopic measurements verified complexatio n, albeit with progressively reduced affinity by more than 1.5 orders of ma gnitude, in mixtures of up to 50% dimethyl sulfoxide (DMSO). Since hevein l acks the compact beta -strand arrangement of other sugar receptors, such a structural motif may confer enhanced resistance to solvent exchange. Two se ttings of solid-phase activity assays proved this assumption for three type s of alpha- and/or beta -galactoside-binding proteins, that is, a human imm unoglobulin G (IgG) subfraction, the mistletoe lectin, and a member of the galectin family of animal lectins. Computer-assisted calculations and NMR e xperiments also revealed no conspicuous impact of the solvent on the confor mational properties of the tested ligands. To define all possible nuclear O verhauser effect (NOE) contacts in a certain conformation and to predict in volvement of exchangeable protons, we established a new screening: protocol applicable during a given molecular dynamics (MD) trajectory and calculate d population densities of distinct contacts. Experimentally, transferred NO E (tr-NOE) experiments with IgG molecules and the disaccharide Gal'alpha1-3 Gal beta1-r in DMSO as; solvent disclosed that such an additional crosspeak that is, Gal'OH2-GalOH4, was even detectable for the bound ligand under co nditions ins which spin diffusion effects are suppressed. Further measureme nts with the plant lectin and galectins confirmed fine broadening of ligand signals and gave access Ito characteristic crosspeaks in the aprotic solve nt and its mixtures with water. Our combined biochemical, computational, an d NMR-spectroscopical strategy is expected to contribute notably to the pre cise elucidation of the geometry of ligands bound to compactly I folded sug ar receptors and of the role of water molecules in protein - ligand (carboh ydrate) recognition, with relevance to areas beyond the glycosciences.