Studying Tight Dimers Using Ordinary Fluorimeter

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00216224%3A14740%2F20%3A00118403" target="_blank" >RIV/00216224:14740/20:00118403 - isvavai.cz</a>

Výsledek na webu

<a href="https://www.ceitec.eu/abstract-book-final-docx-pdf/f36324" target="_blank" >https://www.ceitec.eu/abstract-book-final-docx-pdf/f36324</a>

DOI - Digital Object Identifier

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Jazyk výsledku

angličtina

Název v původním jazyce

Studying Tight Dimers Using Ordinary Fluorimeter

Popis výsledku v původním jazyce

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.

Název v anglickém jazyce

Studying Tight Dimers Using Ordinary Fluorimeter

Popis výsledku anglicky

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.

Druh

O - Ostatní výsledky

CEP obor

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OECD FORD obor

10608 - Biochemistry and molecular biology

Projekt

<a href="/cs/project/LQ1601" target="_blank" >LQ1601: CEITEC 2020</a><br>

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Rok uplatnění

2020

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů