Kinetic and Thermodynamic Evaluation of Kynurenic Acid Binding to GluR1 270−300 Polypeptide by Surface Plasmon Resonance Experiments A ́ da ́ m Juha ́ sz, † Edit Csapó ,* ,†,‡ Ditta Ungor, † Ga ́ bor K. Tó th, ‡ La ́ szló Ve ́ csei, §,∥ and Imre De ́ ka ́ ny* ,†,‡ † MTA-SZTE Supramolecular and Nanostructured Materials Research Group and ‡ Department of Medical Chemistry, Faculty of Medicine, University of Szeged, Dó m té r 8., Szeged H-6720, Hungary § MTA-SZTE Neuroscience Research Group and ∥ Department of Neurology, University of Szeged, Semmelweis u. 6, Szeged H-6725, Hungary ABSTRACT: This work clearly demonstrates an evaluation process that is easily performed and is simply based on the fitting of temperature-dependent surface plasmon resonance (SPR) sensorgrams to provide detailed thermodynamic characterization of biologically relevant interactions. The reversible binding of kynurenic acid (KYNA) on human glutamate receptor (GluR1) polypeptide (GluR1 270−300 )- modified gold surface has been studied at various temperatures under physiological conditions by two-dimensional SPR experiments. The registered sensorgrams were fitted by using different kinetic models without application of any commercial software. Assuming that the association of GluR1 270−300 − KYNA complex is first order in both reactants, the association (k a ) and dissociation (k d ) constants as well as the equilibrium constants (K A ) and the Gibbs free-energy change (ΔG°) were given at 10, 20, 30, and 40 °C. Moreover, the enthalpy (ΔH° = −27.91 kJ mol −1 ), entropy (ΔS° = −60.33 J mol −1 K −1 ), and heat capacity changes (ΔC p = −1.28 kJ mol −1 K −1 ) of the model receptor−ligand system were also calculated using a spreadsheet program. Negative values of ΔG° and ΔH° indicate the exothermic formation of a stable GluR1 270−300 −KYNA complex, because the |ΔH| > |TΔS| relation suggests an enthalpy-driven binding process. The negative ΔH° and ΔS° values strongly support the formation of a salt bridge between KYNA and the positively charged residues of the polypeptide (Arg, Lys) at pH 7.4, confirmed by molecular docking calculations as well. ■ INTRODUCTION Interactions of proteins with small (drug) molecules play a determinant role in living organisms. 1 Detailed kinetic and thermodynamic characterization of receptor−ligand interac- tions may decisively contribute to modern pharmaceutical developments and would be of great benefit to structure-based drug design. 2 A number of different real-time and equilibrium analysis techniques are available that can be used to monitor and quantify protein−ligand association processes. However, radio-labeled assays are reproducible and fast techniques; their major disadvantages are that they are hazardous to human health, produce radioactive waste, and require special laboratory conditions. 3 This has led to the development of “label-free” assays based on optical methods. 4 The two-dimensional (2D) sensor techniques, especially the SPR, are capable of real-time monitoring of these interactions on a gold sensor surface without the use of labels. 5 During SPR measurements, one of the interactants is immobilized from the solution onto a solid/ liquid interface, and a solution of the other interactant is passed over the functionalized gold surface at constant temperature. During this procedure, the refractive index at the interface undergoes a change, this being directly related to the number of biomolecules adsorbed on the surface of the biosensor chip. In addition to the quantitative analysis, the SPR method simultaneously provides kinetic and thermodynamic character- izations of biomolecular interactions. 6 Understanding the interactions between a biological macromolecule and drug molecule requires detailed knowledge of classic physical− chemical parameters. 7 The main objective of this work was to provide important data on the interactions between a model peptide fragment of human glutamate receptor (GluR1 270−300 ) and KYNA by using only SPR experiments. Usually, the fitting of SPR sensorgrams is carried out by using several commercial software programs, but in contrast with these evaluation procedures, we provided the kinetic and thermodynamic data of this model receptor−ligand system via a very simple method. The studied system has relevance in neuroscience. Prescott et al. have published a work about the dual action of KYNA on α- amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (also known as AMPA receptor, AMPAR) responses. 8 In millimolar concentrations, KYNA is an inhibitor of AMPARs, whereas in nanomolar (or micromolar) concentrations, it Received: June 6, 2016 Revised: July 24, 2016 Article pubs.acs.org/JPCB © XXXX American Chemical Society A DOI: 10.1021/acs.jpcb.6b05682 J. Phys. Chem. B XXXX, XXX, XXX−XXX