Publication details
Studying Tight Dimers Using Ordinary Fluorimeter
| Authors | |
|---|---|
| Year of publication | 2020 |
| Type | Conference abstract |
| MU Faculty or unit | |
| Citation | |
| Description | The 14-3-3 proteins represent one part of the 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. In our study, we focused on the zeta isoform with most stable dimers and its phosphorylated form. Using standard biophysical methods we have only seen that the Kd is lower than 1microM. Therefore, we designed very sensitive fluorescence based methods to allow for study of such tighly bound dimers. Using these methods, we determined the dissociation constant to 5 nM, as well as kinetic parameters of the oligomerization process. Moreover, we studied the dependencies of the process on several buffer conditions. Also, we tested the proposed dimer disruption after phosphorylation at Ser58 located at the dimeric interface and measured the Kd and kinetic parameters for the mixed dimer (wildtype - phosphorylated form). Tyrosine hydroxylase is one of many binding partners of 14-3-3 and an enzyme catalyzing the rate-limiting step in the synthesis of catecholamines (dopamine, noradrenaline, adrenaline). We study its regulatory domain that directly interacts with 14-3-3 and thus regulates the function of the whole enzyme. The domain is dimeric and each monomer consists of a structured and an unstructured part of similar size. This considerably restricts the possibilities how to study its structure. We use NMR as it can see with atomic resolution both parts and we can assess the dynamic properties of the domain. We studied the effects of phosphorylation on the structure and the resulting dynamic data for computational studies. |
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