14-3-3 proteins are regulatory proteins involved in many signaling pathways. They play a significant role in the regulation of cell growth, apoptosis, cytoskeletal dynamics and transcription control of genes [1–4]. The 14-3-3 family of proteins consists of seven isoforms in mammals, which interact with a large number of binding partners containing phosphorylated site [5]. The function of 14-3-3 proteins is strongly dependent on their oligomeric state. Detailed characterization of dimer-monomer equilibrium is essential for full understanding of the function of 14-3-3 proteins.
We designed two potential monomeric mutants of 14-3-3ζ to understand monomer-dimer equilibria of 14-3-3ζ protein [6]. Two monomeric variants were firstly examined by standard biophysical methods such as Circular dichroism, Differential scanning calorimetry. The dissociation constant of monomeric mutants was experimentally investigated by NATIVE PAGE and micro-scale thermophoresis. In addition, we experimentally determined dissociation rate constant and dissociation constant of the dimeric form of 14-3-3ζ. In order to achieve this, we designed two fluorescence assays using Förster resonance energy transfer and self-quenching to determine thermodynamic and kinetic parameters of 14-3-3ζ dimerization. Moreover, we studied the stability of 14-3-3ζ dimer under a variety of conditions.
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 the CEITEC 2020 (LQ1601) project. This work was supported by Ministry of Education, Youth and Sports within program INTER-ACTION (LTAUSA18168).