期刊
PROGRESS IN NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY
卷 96, 期 -, 页码 47-57出版社
PERGAMON-ELSEVIER SCIENCE LTD
DOI: 10.1016/j.pnmrs.2016.02.002
关键词
Protein-ligand interaction; Relaxation; Chemical exchange; Chemical shift timescale; Kinetics
资金
- Suntory Holdings
- MEXT, Japan
- Grants-in-Aid for Scientific Research [15H04342] Funding Source: KAKEN
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. (C) 2016 Elsevier B.V. All rights reserved.
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