Expression, purification and crystallization of the
intracellular domains of the ethylene receptor ETR1 from
Arabidopsis thaliana

k 2015

Expression, purification and crystallization of the intracellular domains of the ethylene receptor ETR1 from Arabidopsis thaliana

SZMITKOWSKA, Agnieszka, Zuzana JASEŇÁKOVÁ, Blanka PEKÁROVÁ, Jan KOMÁREK, Lukáš ŽÍDEK et. al.

Basic information

Original name

Expression, purification and crystallization of the intracellular domains of the ethylene receptor ETR1 from Arabidopsis thaliana

Authors

SZMITKOWSKA, Agnieszka, Zuzana JASEŇÁKOVÁ, Blanka PEKÁROVÁ, Jan KOMÁREK, Lukáš ŽÍDEK, Michaela WIMMEROVÁ and Jan HEJÁTKO

Edition

XVII. Setkání biochemiků a molekulárních biologů, 2015

Other information

Language

English

Type of outcome

Prezentace na konferencích

Field of Study

10600 1.6 Biological sciences

Country of publisher

Czech Republic

Confidentiality degree

není předmětem státního či obchodního tajemství

References:

URL

Organization unit

Central European Institute of Technology

ISBN

978-80-210-8015-7

Změněno: 23/9/2016 17:49, Agnieszka Szmitkowska, Ph.D., M.Sc.

Abstract

V originále

ETHYLENE RESPONSE 1 (ETR1) is a membrane-bound receptor from Arabidopsis thaliana involved in ethylene signalling. ETR1 possesses all of the sequence motifs of canonical histidine kinase (HK) domains and also reveals HK activity [1, 2]. HK activity and a presence of a C-terminal receiver domain (RD) may allow for cross-talk between ethylene signalling and multistep phosphorelay (MSP) in Arabidopsis. HK activity is regulated by the presence of divalent ions. Calcium and magnesium (Mg2+) ions are inhibiting HK activity of ETR1. On the other hand manganese (Mn2+) ion causes both HK and serine/threonine activity of ETR1. This mechanism of ETR1 could play a role as a signal switch between canonical CTR1/EIN2/EIN3 cascade of ethylene signalling and MSP pathway in A. thaliana. The main objective of our work is to elucidate structural aspects and HK activity of ETR1 in ethylene/MSP cross-talk. In order to achieve our goal we started with expression of intracellular domains of ETR1 in E. coli expression system and purification using affinity and gel filtration chromatography. We prepared pure, soluble proteins of dimerization, catalytic and receiver domains and we are currently optimising the kinase assay to prove functional activity of these domains. To investigate the structural changes induced by the binding of Mg2+ or Mn2+ ions to RD, X-ray crystallography was used. So far, crystals of RD (soaked or co-crystallized with Mg2+ or Mn2+ ions) were obtained using the procedure described previously [3] and the diffraction data were obtained at 2.5 Å - 2.8 Å resolution. In order to elucidate the effect of ions on RD, crystals diffracting to a better resolution are needed. Further optimization of crystallization conditions (including the temperature, crystallization technique or the presence of additives) is therefore currently ongoing. To overcome the size limits of NMR measurements, we are going to employ intein technology [4]. For that we prepared intein-containing DNA constructs of ETR1 domains. Now we are optimising expression and purification of intein-fused proteins. [1] Gamble et al. (1999), Proc. Natl. Acad. Sci. USA, 95:7825-7829. [2] Moussatche & Klee. (2004), J. Biol. Chem., Vol. 279, No. 47, 48734-48741. [3] Grantz et al. (1998), Acta Crystallogr D Biol Crystallogr. 54, 690-2. [4] Volkmann & Iwai, (2010), Mol. BioSyst., Vol. 6, 2110-2121.

Links

GA13-25280S, research and development project

Name: Elucidating molecular mechanisms of cytokinins-ethylene crosstalk in the plant development

Investor: Czech Science Foundation

Citovat

SZMITKOWSKA, Agnieszka, Zuzana JASEŇÁKOVÁ, Blanka PEKÁROVÁ, Jan KOMÁREK, Lukáš ŽÍDEK, Michaela WIMMEROVÁ and Jan HEJÁTKO. Expression, purification and crystallization of the intracellular domains of the ethylene receptor ETR1 from Arabidopsis thaliana. In XVII. Setkání biochemiků a molekulárních biologů. 2015. ISBN 978-80-210-8015-7.

@proceedings{1354848,
   author = {Szmitkowska, Agnieszka and Jaseňáková, Zuzana and Pekárová, Blanka and Komárek, Jan and Žídek, Lukáš and Wimmerová, Michaela and Hejátko, Jan},
   booktitle = {XVII. Setkání biochemiků a molekulárních biologů},
   language = {eng},
   isbn = {978-80-210-8015-7},
   title = {Expression, purification and crystallization of the intracellular domains of the ethylene receptor ETR1 from Arabidopsis thaliana},
   url = {http://orion.chemi.muni.cz/setkani/Setkani17/index.htm},
   year = {2015}
}

TY  - CONF
ID  - 1354848
AU  - Szmitkowska, Agnieszka - Jaseňáková, Zuzana - Pekárová, Blanka - Komárek, Jan - Žídek, Lukáš - Wimmerová, Michaela - Hejátko, Jan
PY  - 2015
TI  - Expression, purification and crystallization of the intracellular domains of the ethylene receptor ETR1 from Arabidopsis thaliana
SN  - 9788021080157
UR  - http://orion.chemi.muni.cz/setkani/Setkani17/index.htm
N2  - ETHYLENE RESPONSE 1 (ETR1) is a membrane-bound receptor from Arabidopsis thaliana involved in ethylene signalling. ETR1 possesses all of the sequence motifs of canonical histidine kinase (HK) domains and also reveals HK activity [1, 2]. HK activity and a presence of a C-terminal receiver domain (RD) may allow for cross-talk between ethylene signalling and multistep phosphorelay (MSP) in Arabidopsis. HK activity is regulated by the presence of divalent ions. Calcium and magnesium (Mg2+) ions are inhibiting HK activity of ETR1. On the other hand manganese (Mn2+) ion causes both HK and serine/threonine activity of ETR1. This mechanism of ETR1 could play a role as a signal switch between canonical CTR1/EIN2/EIN3 cascade of ethylene signalling and MSP pathway in A. thaliana. The main objective of our work is to elucidate structural aspects and HK activity of ETR1 in ethylene/MSP cross-talk. In order to achieve our goal we started with expression of intracellular domains of ETR1 in E. coli expression system and purification using affinity and gel filtration chromatography. We prepared pure, soluble proteins of dimerization, catalytic and receiver domains and we are currently optimising the kinase assay to prove functional activity of these domains. To investigate the structural changes induced by the binding of Mg2+ or Mn2+ ions to RD, X-ray crystallography was used. So far, crystals of RD (soaked or co-crystallized with Mg2+ or Mn2+ ions) were obtained using the procedure described previously [3] and the diffraction data were obtained at 2.5 Å - 2.8 Å resolution. In order to elucidate the effect of ions on RD, crystals diffracting to a better resolution are needed. Further optimization of crystallization conditions (including the temperature, crystallization technique or the presence of additives) is therefore currently ongoing. To overcome the size limits of NMR measurements, we are going to employ intein technology [4]. For that we prepared intein-containing DNA constructs of ETR1 domains. Now we are optimising expression and purification of intein-fused proteins. [1] Gamble et al. (1999), Proc. Natl. Acad. Sci. USA, 95:7825-7829. [2] Moussatche & Klee. (2004), J. Biol. Chem., Vol. 279, No. 47, 48734-48741. [3] Grantz et al. (1998), Acta Crystallogr D Biol Crystallogr. 54, 690-2. [4] Volkmann & Iwai, (2010), Mol. BioSyst., Vol. 6, 2110-2121.
ER  -

SZMITKOWSKA, Agnieszka, Zuzana JASEŇÁKOVÁ, Blanka PEKÁROVÁ, Jan KOMÁREK, Lukáš ŽÍDEK, Michaela WIMMEROVÁ and Jan HEJÁTKO. Expression, purification and crystallization of the intracellular domains of the ethylene receptor ETR1~from~Arabidopsis thaliana. In \textit{XVII. Setkání biochemiků a molekulárních biologů}. 2015. ISBN~978-80-210-8015-7.

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