Studying Tight Dimers Using Ordinary Fluorimeter

a 2020

Studying Tight Dimers Using Ordinary Fluorimeter

LOUŠA, Petr a Jozef HRITZ

Základní údaje

Originální název

Studying Tight Dimers Using Ordinary Fluorimeter

Autoři

LOUŠA, Petr (203 Česká republika, domácí) a Jozef HRITZ (703 Slovensko, domácí)

Vydání

CEITEC PhD Conference, 2020

Další údaje

Jazyk

angličtina

Typ výsledku

Konferenční abstrakt

Obor

10608 Biochemistry and molecular biology

Stát vydavatele

Česká republika

Utajení

není předmětem státního či obchodního tajemství

Odkazy

URL

Kód RIV

RIV/00216224:14740/20:00118403

Organizační jednotka

Středoevropský technologický institut

Klíčová slova anglicky

Ordinary Fluorimeter

Štítky

rivok

Změněno: 17. 3. 2021 14:29, Mgr. Pavla Foltynová, Ph.D.

Anotace

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.

Návaznosti

LQ1601, projekt VaV

Název: CEITEC 2020 (Akronym: CEITEC2020)

Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, CEITEC 2020

Citovat

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

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