14-3-3 proteins are regulatory proteins involved in many signaling pathways. They play a key role in nervous system and neurodegeneration [1–3]. The 14-3-3 family of proteins consists of seven isoforms in mammals, which interact with large number of binding partners containing phosphorylated Serine or Threonine [4]. The X-ray three-dimensional structure showed dimeric form of 14‑3‑3. Each monomer consists of 9 α‑helices in an antiparallel arrangement. Dimer interface is stabilized by multiple conserved hydrophobic interactions (e.g. L12), polar contacts and by several isoform-specific salt bridges (e.g. K78) [5–6]. However, dimer dissociation constants as well as fundamental kinetic rate constants remain unknown.
In order to study thermodynamics and kinetics of 14-3-3ζ dimer, we prepared a new construct with a single accessible cysteine at the N terminus of protein. This construct was used in our recently developed assays based on the Förster resonance energy transfer (FRET) and self-quenching (SQ) phenomena and in microscale thermophoresis (MST) methodology. We determined the thermodynamic parameter (dissociation constant, Kd) and kinetic parameter represented by life-time of 14-3-3ζ dimer. Moreover, we studied the stability of 14-3-3ζ dimer under variety of factors.
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.