Quantitative analysis of protein–ligand interactions by NMR

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Quantitative analysis of protein–ligand interactions by NMR

Publication date: Available online 3 March 2016
Source:Progress in Nuclear Magnetic Resonance Spectroscopy

Author(s): Ayako Furukawa, Tsuyoshi Konuma, Saeko Yanaka, Kenji Sugase

Protein–ligand interactions have been commonly studied through static structures of the protein–ligand complex. Recently, however, there has been increasing interest in investigating the dynamics of protein–ligand interactions both for fundamental understanding of the underlying mechanisms and for drug development. NMR is a versatile and powerful tool, especially because it provides site-specific quantitative information. NMR has widely been used to determine the dissociation constant (K D), in particular, for relatively weak interactions. The simplest NMR method is a chemical-shift titration experiment, in which the chemical-shift changes of a protein in response to ligand titration are measured. There are other quantitative NMR methods, but they mostly apply only to interactions in the fast-exchange regime. These methods derive the dissociation constant from population-averaged NMR quantities of the free and bound states of a protein or ligand. In contrast, the recent advent of new relaxation-based experiments, including R 2 relaxation dispersion and ZZ-exchange, has enabled us to obtain kinetic information on protein–ligand interactions in the intermediate- and slow-exchange regimes. Based on R 2 dispersion or ZZ-exchange, methods that can determine the association rate, k on, dissociation rate, k off, and K D have been developed. In these approaches, R 2 dispersion or ZZ-exchange curves are measured for multiple samples with different protein and/or ligand concentration ratios, and the relaxation data are fitted to theoretical kinetic models. It is critical to choose an appropriate kinetic model, such as the two- or three-state exchange model, to derive the correct kinetic information. The R 2 dispersion and ZZ-exchange methods are suitable for the analysis of protein–ligand interactions with a micromolar or sub-micromolar dissociation constant but not for very weak interactions, which are typical in very fast exchange. This contrasts with the NMR methods that are used to analyze population-averaged NMR quantities. Essentially, to apply NMR successfully, both the type of experiment and equation to fit the data must be carefully and specifically chosen for the protein–ligand interaction under analysis. In this review, we first explain the exchange regimes and kinetic models of protein–ligand interactions, and then describe the NMR methods that quantitatively analyze these specific interactions.
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