NMR-based analysis of protein–ligand interactions

Abstract

Physiological processes are mainly controlled by intermolecular recognition mechanisms involving protein-protein and protein-ligand (low molecular weight molecules) interactions. One of the most important tools for probing these interactions is high-field solution nuclear magnetic resonance (NMR) through protein-observed and ligand-observed experiments, where the protein receptor or the organic compounds are selectively detected. NMR binding experiments rely on comparison of NMR parameters of the free and bound states of the molecules. Ligand-observed methods are not limited by the protein molecular size and therefore have great applicability for analysing protein-ligand interactions. The use of these NMR techniques has considerably expanded in recent years, both in chemical biology and in drug discovery. We review here three major ligand-observed NMR methods that depend on the nuclear Overhauser effect-transferred nuclear Overhauser effect spectroscopy, saturation transfer difference spectroscopy and water-ligand interactions observed via gradient spectroscopy experiments-with the aim of reporting recent developments and applications for the characterization of protein-ligand complexes, including affinity measurements and structural determination.

Computational allosteric ligand binding site identification on Ras proteins

“…[92,109]). The notion that the ligand binding potential of a site is proportional to the frequency of probe binding to 'hotspot' residues also forms the basis for fragment screening by NMR [119,120], where for example, difference spectra of an N 15 -labeled protein before and after the addition of molecular probes can be used to determine the ligand binding potential of a site [119]. Similarly, fragment-based computational ligand screening methods such as SEED [84] rely on docking of comparatively few chemical building blocks derived from drug-like molecules to serve as scaffolds for the construction of novel ligands [120].…”

Drug Discovery and Development: the Role of NMR

“…17 There are several attractive features to these methods including (i) the ability to screen ligands for binding interactions to a target protein in mixtures, improving the screening efficiency, (ii) the ability to screen at relatively low protein concentration, thus limiting protein reagent supply issues, (iii) the ability to screen the protein without the need for isotope labeling, which can be time consuming and expensive, (iv) the ability to derive at least some structural information on the mode of binding, and (v) the ability to derive binding constants directly from these NMR experiments. The alternative approach is to titrate mixtures of ligands into a solution of an isotopically labeled protein and observe changes in the chemical shifts of protein resonances in contact with ligand molecules in the binding site.…”

Le criblage de fragments

“…Les expériences sont généralement réalisées à haute concentration de fragments (> 100 M) dans une solution aqueuse tamponnée [2,6,7]. La RMN s'avère particulièrement efficace pour détecter des interactions entre les petites molécules (les fragments) et les cibles protéines ou nucléiques [8,9]. On distingue deux types d'expériences, selon que le spectre RMN des molécules est directement observé pour détec-ter des interactions, ou que le spectre de la cible thé-rapeutique est comparé en l'absence et en présence de fragments.…”