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Unraveling Complex Small‐Molecule Binding Mechanisms by Using Simple NMR Spectroscopy
Author(s) -
Quinternet Marc,
Starck JeanPhilippe,
Delsuc MarcAndré,
Kieffer Bruno
Publication year - 2012
Publication title -
chemistry – a european journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.687
H-Index - 242
eISSN - 1521-3765
pISSN - 0947-6539
DOI - 10.1002/chem.201101983
Subject(s) - heteronuclear molecule , chemistry , dissociation constant , isothermal titration calorimetry , titration , ligand (biochemistry) , protein ligand , nuclear magnetic resonance spectroscopy , dissociation (chemistry) , molecule , small molecule , stereochemistry , biochemistry , organic chemistry , receptor
Heteronuclear NMR spectroscopy provides a unique way to obtain site‐specific information about protein–ligand interactions. Usually, such studies rely on the availability of isotopically labeled proteins, thereby allowing both editing of the spectra and ligand signals to be filtered out. Herein, we report that the use of the methyl SOFAST correlation experiment enables the determination of site‐specific equilibrium binding constants by using unlabeled proteins. By using the binding of L ‐ and D ‐tryptophan to serum albumin as a test case, we determined very accurate dissociation constants for both the high‐ and low‐affinity sites present at the protein surface. The values of site‐specific dissociation constants were closer to those obtained by isothermal titration calorimetry than those obtained from ligand‐observed methods, such as saturation transfer difference. The possibility of measuring ligand binding to serum albumin at physiological concentrations with unlabeled proteins may open up new perspectives in the field of drug discovery.