title: Quantitative analysis of protein-ligand interactions by NMR.
abstract: 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 (KD), 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 R2 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 R2 dispersion or ZZ-exchange, methods that can determine the association rate, kon, dissociation rate, koff, and KD have been developed. In these approaches, R2 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 R2 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.
authors: Bioorganic Research Institute, Suntory Foundation for Life Sciences, 1-1-1 Wakayamadai, Shimamoto, Mishima, Osaka 618-8503, Japan; Graduate School of Medical Life Science, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan.
authors: Ayako
authors: A
authors: Furukawa
authors: Bioorganic Research Institute, Suntory Foundation for Life Sciences, 1-1-1 Wakayamadai, Shimamoto, Mishima, Osaka 618-8503, Japan; Department of Structural and Chemical Biology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
authors: Tsuyoshi
authors: T
authors: Konuma
authors: Bioorganic Research Institute, Suntory Foundation for Life Sciences, 1-1-1 Wakayamadai, Shimamoto, Mishima, Osaka 618-8503, Japan; Department of Life and Coordination-Complex Molecular Science, Biomolecular Functions, Institute of Molecular Science, National Institute of Natural Sciences, Japan.
authors: Saeko
authors: S
authors: Yanaka
authors: Bioorganic Research Institute, Suntory Foundation for Life Sciences, 1-1-1 Wakayamadai, Shimamoto, Mishima, Osaka 618-8503, Japan; Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto-Daigaku Katsura, Nishikyo-Ku, Kyoto 615-8510, Japan. Electronic address: sugase@moleng.kyoto-u.ac.jp.
authors: Kenji
authors: K
authors: Sugase
mesh_terms: D055592:Biophysical Phenomena; D007700:Kinetics; D008024:Ligands; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011485:Protein Binding; D011506:Proteins / Q000032:analysis* / Q000737:chemistry*
chemical_list: D008024:Ligands; D011506:Proteins
keywords: Chemical exchange; Chemical shift timescale; Kinetics; Protein–ligand interaction; Relaxation