Large number of proteins are forming homodimers. Here, we are presenting experimental and computational tools for the characterization of stable homodimers in terms of biophysical and structural properties of the dimer dissociation. First, fluorescence assays analyzed by ad-hoc mathematical models as presented here allow efficient and reliable determination of thermodynamic and kinetic parameters of dimer-monomer equilibria. To quantify microscopic dynamics between monomers and homodimers, we have designed sensitive fluorescent assays based on the Förster resonance energy transfer (FRET) and self-quenching (SQ) phenomena. The applicability of these approaches is shown here for the determination of dissociation constant (Kd) and dissociation and association rate constants (koff and kon, respectively) of 14-3-3ζ dimer-monomer equilibria. The most important biophysical factors altering this equilibrium are presented here.
Second, computational approach of Hamiltonian replica exchange molecular dynamics (H-REMD) combined with the distance restraints between the monomers is applied to reveal the structural details of the homodimer dissociation/association pathway. Its applicability is presented here for the case of the regulatory domain of human tyrosine hydroxylase 1 (RD-hTH1). Next, we analyzed the free energy profile and calculated the binding affinities, and compared the computational results with the experimental observations for three prepared human RD-hTH1 constructs.
This work was supported by the research grant from the Czech Science Foundation, grant no. GA. 15-34684L. The results of this research have been acquired within CEITEC 2020 (LQ1601) project.