Probing the Kinetic Landscape of Transient Peptide−Protein Interactions by Use of Peptide ¹⁵N NMR Relaxation Dispersion Spectroscopy:  Binding of an Antithrombin Peptide to Human Prothrombin

Abstract

Protein-ligand interactions may lead to the formation of multiple molecular complexes in dynamic exchange, affecting the kinetic and thermodynamic characteristics of the binding equilibrium. We followed the dissociation kinetics of the transient and specific complex of an antithrombotic peptide N-acetyl-Asp(55)-Phe-Glu-Glu-Ile-Pro(60)-Glu-Glu-Tyr-Leu-Gln(65) with human prothrombin by use of (15)N NMR relaxation dispersion spectroscopy of the peptide. Every one of the five (15)N-labeled adjacent residues of the peptide exhibited apparently different kinetic exchange and relaxation behaviors, which were especially evident at different concentrations of prothrombin. Binding-induced (15)N relaxation dispersion of residues Phe(56), Glu(57), Glu(58), and Ile(59) can be fitted phenomenologically to a two-site on-and-off exchange mechanism with physically feasible relaxation and kinetic parameters obtained for residues Phe(56), Glu(58), and Ile(59), independent of the prothrombin concentration. The apparent kinetic parameters of Glu(57) show some dependence on the concentration of prothrombin and the extracted transverse relaxation rate for Glu(57) in the bound state was severalfold higher than that expected for a protein-peptide complex with a size of approximately 72 kDa. In addition, the equilibrium population of the bound peptide obtained for Glu(57) was inconsistent with those for Phe(56), Glu(58), and Ile(59) and with the prothrombin/peptide ratios used in the experiments. These discrepancies can be explained by the presence of two conformations for the peptide-protein complex exchanging at a rate of approximately 100 s(-)(1). In all, our study shows that fast dissociation of protein-peptide complexes can be studied quantitatively using peptide (15)N NMR relaxation dispersion measurements without a precise knowledge of the peptide and protein concentrations. In addition, protein titration was found to improve the accuracy of quantitative analysis and may make it possible to determine the rate of conformational changes within the protein-peptide complex.

Novel Insights into the Mechanisms of CIN85 SH3 Domains Binding to Cbl Proteins: Solution-Based Investigations and In Vivo Implications

“…Indeed, at intermediate concentrations, the binding rates may be affected, hence indicating the presence of other conformational states of the interacting residues. 33 Titration of CIN85A with Cbl-b(E) produced the same unusual curved CS changes for residues as in Cbl-b titration; however, Cbl-b(F) abrogates this effect (Fig. 5).…”

Alternate Binding Modes for a Ubiquitin–SH3 Domain Interaction Studied by NMR Spectroscopy

“…[24][25][26] This situation leads to exchange line broadening that is undesired in titration experiments and in experiments that are used for structure determination. Contributions to linewidths of resonances from conformational exchange on the millisecond time scale can be quantified by relaxation dispersion NMR, providing detailed information on the exchange process.…”

Protein and RNA Dynamics Play Key Roles in Determining the Specific Recognition of GU-rich Polyadenylation Regulatory Elements by Human Cstf-64 Protein

“…33,34 Effective transverse relaxation rate constants, R eff 2 , were measured as a function of the field strength, y CP , where y CP Z 1=4t CP and 2t CP is the time between the centers of successive 1808 pulses in the CPMG sequence. Figure 7 shows representative 15 N relaxation dispersion data of the backbone amides.…”