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A New Combined Computational and NMR‐Spectroscopical Strategy for the Identification of Additional Conformational Constraints of the Bound Ligand in an Aprotic Solvent
Author(s) -
Siebert HansChristian,
André Sabine,
Asensio Juan Luis,
Cañada Francisco Javier,
Dong Xin,
Espinosa Juan Felix,
Frank Martin,
Gilleron Martine,
Kaltner Herbert,
Kozár Tibor,
Bovin Nicolai V.,
von der Lieth ClausWilhelm,
Vliegenthart Johannes F. G.,
JiménezBarbero Jesús,
Gabius HansJoachim
Publication year - 2000
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/1439-7633(20001002)1:3<181::aid-cbic181>3.0.co;2-9
Subject(s) - chemistry , solvent , ligand (biochemistry) , molecule , proton nmr , molecular dynamics , lectin , solvent effects , two dimensional nuclear magnetic resonance spectroscopy , receptor , stereochemistry , computational chemistry , organic chemistry , biochemistry
This study documents the feasibility of switching to an aprotic medium in sugar receptor research. The solvent change offers additional insights into mechanistic details of receptor–carbohydrate ligand interactions. If a receptor retained binding capacity in an aprotic medium, solvent‐exchangeable protons of the ligand would not undergo transfer and could act as additional sensors, thus improving the level of reliability in conformational analysis. To probe this possibility, we first focused on hevein, the smallest lectin found in nature. The NMR‐spectroscopic measurements verified complexation, albeit with progressively reduced affinity by more than 1.5 orders of magnitude, in mixtures of up to 50 % dimethyl sulfoxide (DMSO). Since hevein lacks the compact β ‐strand arrangement of other sugar receptors, such a structural motif may confer enhanced resistance to solvent exchange. Two settings of solid‐phase activity assays proved this assumption for three types of α ‐ and/or β ‐galactoside‐binding proteins, that is, a human immunoglobulin G (IgG) subfraction, the mistletoe lectin, and a member of the galectin family of animal lectins. Computer‐assisted calculations and NMR experiments also revealed no conspicuous impact of the solvent on the conformational properties of the tested ligands. To define all possible nuclear Overhauser effect (NOE) contacts in a certain conformation and to predict involvement of exchangeable protons, we established a new screening protocol applicable during a given molecular dynamics (MD) trajectory and calculated population densities of distinct contacts. Experimentally, transferred NOE (tr‐NOE) experiments with IgG molecules and the disaccharide Gal′ α 1‐3Gal β 1‐R in DMSO as solvent disclosed that such an additional crosspeak, that is, Gal′ OH2–Gal OH4, was even detectable for the bound ligand under conditions in which spin diffusion effects are suppressed. Further measurements with the plant lectin and galectins confirmed line broadening of ligand signals and gave access to characteristic crosspeaks in the aprotic solvent and its mixtures with water. Our combined biochemical, computational, and NMR‐spectroscopical strategy is expected to contribute notably to the precise elucidation of the geometry of ligands bound to compactly folded sugar receptors and of the role of water molecules in protein–ligand (carbohydrate) recognition, with relevance to areas beyond the glycosciences.