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A homogeneous quenching resonance energy transfer assay for the kinetic analysis of the GTPase nucleotide exchange reaction

Standard

A homogeneous quenching resonance energy transfer assay for the kinetic analysis of the GTPase nucleotide exchange reaction. / Kopra, Kari; Ligabue, Alessio; Wang, Qi; Syrjänpää, Markku; Blaževitš, Olga; Veltel, Stefan; van Adrichem, Arjan J; Hänninen, Pekka; Abankwa, Daniel; Härmä, Harri.

in: ANAL BIOANAL CHEM, Jahrgang 406, Nr. 17, 01.07.2014, S. 4147-56.

Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung

Harvard

Kopra, K, Ligabue, A, Wang, Q, Syrjänpää, M, Blaževitš, O, Veltel, S, van Adrichem, AJ, Hänninen, P, Abankwa, D & Härmä, H 2014, 'A homogeneous quenching resonance energy transfer assay for the kinetic analysis of the GTPase nucleotide exchange reaction', ANAL BIOANAL CHEM, Jg. 406, Nr. 17, S. 4147-56. https://doi.org/10.1007/s00216-014-7795-7

APA

Kopra, K., Ligabue, A., Wang, Q., Syrjänpää, M., Blaževitš, O., Veltel, S., van Adrichem, A. J., Hänninen, P., Abankwa, D., & Härmä, H. (2014). A homogeneous quenching resonance energy transfer assay for the kinetic analysis of the GTPase nucleotide exchange reaction. ANAL BIOANAL CHEM, 406(17), 4147-56. https://doi.org/10.1007/s00216-014-7795-7

Vancouver

Kopra K, Ligabue A, Wang Q, Syrjänpää M, Blaževitš O, Veltel S et al. A homogeneous quenching resonance energy transfer assay for the kinetic analysis of the GTPase nucleotide exchange reaction. ANAL BIOANAL CHEM. 2014 Jul 1;406(17):4147-56. https://doi.org/10.1007/s00216-014-7795-7

Bibtex

@article{f057666f9c1043338905ae6c38cf233e,
title = "A homogeneous quenching resonance energy transfer assay for the kinetic analysis of the GTPase nucleotide exchange reaction",
abstract = "A quenching resonance energy transfer (QRET) assay for small GTPase nucleotide exchange kinetic monitoring is demonstrated using nanomolar protein concentrations. Small GTPases are central signaling proteins in all eukaryotic cells acting as a {"}molecular switches{"} that are active in the GTP-state and inactive in the GDP-state. GTP-loading is highly regulated by guanine nucleotide exchange factors (GEFs). In several diseases, most prominently cancer, this process in misregulated. The kinetics of the nucleotide exchange reaction reports on the enzymatic activity of the GEF reaction system and is, therefore, of special interest. We determined the nucleotide exchange kinetics using europium-labeled GTP (Eu-GTP) in the QRET assay for small GTPases. After GEF catalyzed GTP-loading of a GTPase, a high time-resolved luminescence signal was found to be associated with GTPase bound Eu-GTP, whereas the non-bound Eu-GTP fraction was quenched by soluble quencher. The association kinetics of the Eu-GTP was measured after GEF addition, whereas the dissociation kinetics could be determined after addition of unlabeled GTP. The resulting association and dissociation rates were in agreement with previously published values for H-Ras(Wt), H-Ras(Q61G), and K-Ras(Wt), respectively. The broader applicability of the QRET assay for small GTPases was demonstrated by determining the kinetics of the Ect2 catalyzed RhoA(Wt) GTP-loading. The QRET assay allows the use of nanomolar protein concentrations, as more than 3-fold signal-to-background ratio was achieved with 50 nM GTPase and GEF proteins. Thus, small GTPase exchange kinetics can be efficiently determined in a HTS compatible 384-well plate format.",
keywords = "Animals, Bioluminescence Resonance Energy Transfer Techniques, Energy Transfer, GTP Phosphohydrolases, Humans, Nucleotides",
author = "Kari Kopra and Alessio Ligabue and Qi Wang and Markku Syrj{\"a}np{\"a}{\"a} and Olga Bla{\v z}evit{\v s} and Stefan Veltel and {van Adrichem}, {Arjan J} and Pekka H{\"a}nninen and Daniel Abankwa and Harri H{\"a}rm{\"a}",
year = "2014",
month = jul,
day = "1",
doi = "10.1007/s00216-014-7795-7",
language = "English",
volume = "406",
pages = "4147--56",
journal = "ANAL BIOANAL CHEM",
issn = "1618-2642",
publisher = "Springer",
number = "17",

}

RIS

TY - JOUR

T1 - A homogeneous quenching resonance energy transfer assay for the kinetic analysis of the GTPase nucleotide exchange reaction

AU - Kopra, Kari

AU - Ligabue, Alessio

AU - Wang, Qi

AU - Syrjänpää, Markku

AU - Blaževitš, Olga

AU - Veltel, Stefan

AU - van Adrichem, Arjan J

AU - Hänninen, Pekka

AU - Abankwa, Daniel

AU - Härmä, Harri

PY - 2014/7/1

Y1 - 2014/7/1

N2 - A quenching resonance energy transfer (QRET) assay for small GTPase nucleotide exchange kinetic monitoring is demonstrated using nanomolar protein concentrations. Small GTPases are central signaling proteins in all eukaryotic cells acting as a "molecular switches" that are active in the GTP-state and inactive in the GDP-state. GTP-loading is highly regulated by guanine nucleotide exchange factors (GEFs). In several diseases, most prominently cancer, this process in misregulated. The kinetics of the nucleotide exchange reaction reports on the enzymatic activity of the GEF reaction system and is, therefore, of special interest. We determined the nucleotide exchange kinetics using europium-labeled GTP (Eu-GTP) in the QRET assay for small GTPases. After GEF catalyzed GTP-loading of a GTPase, a high time-resolved luminescence signal was found to be associated with GTPase bound Eu-GTP, whereas the non-bound Eu-GTP fraction was quenched by soluble quencher. The association kinetics of the Eu-GTP was measured after GEF addition, whereas the dissociation kinetics could be determined after addition of unlabeled GTP. The resulting association and dissociation rates were in agreement with previously published values for H-Ras(Wt), H-Ras(Q61G), and K-Ras(Wt), respectively. The broader applicability of the QRET assay for small GTPases was demonstrated by determining the kinetics of the Ect2 catalyzed RhoA(Wt) GTP-loading. The QRET assay allows the use of nanomolar protein concentrations, as more than 3-fold signal-to-background ratio was achieved with 50 nM GTPase and GEF proteins. Thus, small GTPase exchange kinetics can be efficiently determined in a HTS compatible 384-well plate format.

AB - A quenching resonance energy transfer (QRET) assay for small GTPase nucleotide exchange kinetic monitoring is demonstrated using nanomolar protein concentrations. Small GTPases are central signaling proteins in all eukaryotic cells acting as a "molecular switches" that are active in the GTP-state and inactive in the GDP-state. GTP-loading is highly regulated by guanine nucleotide exchange factors (GEFs). In several diseases, most prominently cancer, this process in misregulated. The kinetics of the nucleotide exchange reaction reports on the enzymatic activity of the GEF reaction system and is, therefore, of special interest. We determined the nucleotide exchange kinetics using europium-labeled GTP (Eu-GTP) in the QRET assay for small GTPases. After GEF catalyzed GTP-loading of a GTPase, a high time-resolved luminescence signal was found to be associated with GTPase bound Eu-GTP, whereas the non-bound Eu-GTP fraction was quenched by soluble quencher. The association kinetics of the Eu-GTP was measured after GEF addition, whereas the dissociation kinetics could be determined after addition of unlabeled GTP. The resulting association and dissociation rates were in agreement with previously published values for H-Ras(Wt), H-Ras(Q61G), and K-Ras(Wt), respectively. The broader applicability of the QRET assay for small GTPases was demonstrated by determining the kinetics of the Ect2 catalyzed RhoA(Wt) GTP-loading. The QRET assay allows the use of nanomolar protein concentrations, as more than 3-fold signal-to-background ratio was achieved with 50 nM GTPase and GEF proteins. Thus, small GTPase exchange kinetics can be efficiently determined in a HTS compatible 384-well plate format.

KW - Animals

KW - Bioluminescence Resonance Energy Transfer Techniques

KW - Energy Transfer

KW - GTP Phosphohydrolases

KW - Humans

KW - Nucleotides

U2 - 10.1007/s00216-014-7795-7

DO - 10.1007/s00216-014-7795-7

M3 - SCORING: Journal article

C2 - 24760397

VL - 406

SP - 4147

EP - 4156

JO - ANAL BIOANAL CHEM

JF - ANAL BIOANAL CHEM

SN - 1618-2642

IS - 17

ER -