The 14-3-3 proteins represent a large group of dimeric proteins. Specifically, the 14-3-3 family consists of 7 isoforms, that can form many homo- and heterodimeric states, not even accounting for the possibility of changing the oligomerization properties by posttranslational modifications such as phosphorylation. The functions of 14-3-3 are very often dependent on the dimeric state. Therefore, the parameters of oligomerization are very interesting in order to correctly understand the regulation of 14-3-3 itself.
In our study, we focused on the zeta isoform (most abundant isoform in human brain, also forming most stable dimers) and its phosphorylated form. Using standard biophysical methods we have only seen that the Kd is lower than 1 μM. Therefore, we designed very sensitive fluorescence based methods to allow for study of such tightly bound dimers. Using these methods, we determined the dissociation constant, as well as kinetic parameters of the oligomerization process. Moreover, we studied the dependencies of the process on several buffer conditions, including temperature and ionic strength.
In order to determine the effect of phosphorylation on Ser58, which the literature is not sure about, we used our fluorescence assays. The serine S58 is located at the dimeric interface and therefore its phosphorylation affects the dimerization process. We succeeded in determination of both kinetic and equilibrium parameters of the interaction between non-phosphorylated and phosphorylated 14-3-3ζ protein.
This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic within programme INTER-ACTION (project No. LTAUSA18168) and by the research grant from the Czech Science Foundation, grant no. GA15-34684L and GF20-05789L. Research infrastructure was supported by CEITEC 2020 (LQ1601) and the CIISB research infrastructure project supported by MEYS CR (LM2018127).