Studying Tight Dimers Using Ordinary Fluorimeter

a 2020

Studying Tight Dimers Using Ordinary Fluorimeter

LOUŠA, Petr and Jozef HRITZ

Basic information

Original name

Studying Tight Dimers Using Ordinary Fluorimeter

Authors

LOUŠA, Petr (203 Czech Republic, belonging to the institution) and Jozef HRITZ (703 Slovakia, belonging to the institution)

Edition

CEITEC PhD Conference, 2020

Other information

Language

English

Type of outcome

Konferenční abstrakt

Field of Study

10608 Biochemistry and molecular biology

Country of publisher

Czech Republic

Confidentiality degree

není předmětem státního či obchodního tajemství

References:

URL

RIV identification code

RIV/00216224:14740/20:00118403

Organization unit

Central European Institute of Technology

Keywords in English

Ordinary Fluorimeter

Abstract

V originále

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.

Links

LQ1601, research and development project

Name: CEITEC 2020 (Acronym: CEITEC2020)

Investor: Ministry of Education, Youth and Sports of the CR

Citovat

LOUŠA, Petr and Jozef HRITZ. Studying Tight Dimers Using Ordinary Fluorimeter. In CEITEC PhD Conference. 2020.

@proceedings{1753025,
   author = {Louša, Petr and Hritz, Jozef},
   booktitle = {CEITEC PhD Conference},
   keywords = {Ordinary Fluorimeter},
   language = {eng},
   title = {Studying Tight Dimers Using Ordinary Fluorimeter},
   url = {https://www.ceitec.eu/abstract-book-final-docx-pdf/f36324},
   year = {2020}
}

TY  - CONF
ID  - 1753025
AU  - Louša, Petr - Hritz, Jozef
PY  - 2020
TI  - Studying Tight Dimers Using Ordinary Fluorimeter
KW  - Ordinary Fluorimeter
UR  - https://www.ceitec.eu/abstract-book-final-docx-pdf/f36324
N2  - 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.
ER  -

LOUŠA, Petr and Jozef HRITZ. Studying Tight Dimers Using Ordinary Fluorimeter. In \textit{CEITEC PhD Conference}. 2020.

Detailed Information on Publication Record