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

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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-RasWt, H-RasQ61G, and K-RasWt, respectively. The broader applicability of the QRET assay for small GTPases was demonstrated by determining the kinetics of the Ect2 catalyzed RhoAWt 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.

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Abbreviations

ACN:

Acetonitrile

AHC-spacer:

Aminohexylcarbamoyl-spacer

CV%:

Coefficient of variation

EDTA:

Ethylenediaminetetraacetic acid

GAP:

GTPase-activating protein

GDP:

Guanosine diphosphate

GEF:

Guanine nucleotide exchange factor

Gln:

Glutamine

Gly:

Glycine

GTP:

Guanosine triphosphate

HTS:

High throughput screening

ITMS:

Ion trap mass spectrometer

Kd :

Dissociation constant

kobs :

Observed association rate

koff :

Dissociation rate

kon :

Association rate

Ln3+ :

Lanthanide

LOD:

Limit-of-detection

mant:

Methylanthraniloyl

NMR:

Nuclear magnetic resonance

Q2:

Quench II

QRET:

Quenching resonance energy transfer

S/B:

Signal-to-background ratio

SD:

Standard deviation

TEAAc:

Triethylamine acetic acid

TRL:

Time-resolved luminescence

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Acknowledgments

This work was supported by the Academy of Finland (138584), FP7 Collaborative Project, NANOGNOSTICS (HEALT-F5-2009-242264), Feodor Lynen Return Fellowship from the Alexander von Humboldt-foundation, and the National Doctoral program in Informational and Structural Biology.

The authors declare the following competing financial interest(s): Harri Härmä has commercial interest to the Quench II molecule through QRET Technologies.

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Authors and Affiliations

  1. Institute of Biomedicine, Department of Cell Biology and Anatomy, Laboratory of Biophysics, University of Turku, Tykistökatu 6A 5th floor, 20520, Turku, Finland

    Kari Kopra, Qi Wang, Markku Syrjänpää, Pekka Hänninen & Harri Härmä

  2. Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, 20520, Turku, Finland

    Alessio Ligabue, Olga Blaževitš & Daniel Abankwa

  3. University Medical Center Hamburg-Eppendorf, Martinistraße 52, 20246, Hamburg, Germany

    Stefan Veltel

  4. Institute for Molecular Medicine Finland, University of Helsinki, 00014, Helsinki, Finland

    Arjan J. van Adrichem

Authors
  1. Kari Kopra
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  2. Alessio Ligabue
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  3. Qi Wang
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  4. Markku Syrjänpää
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Correspondence to Kari Kopra.

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Kopra, K., Ligabue, A., Wang, Q. et al. A homogeneous quenching resonance energy transfer assay for the kinetic analysis of the GTPase nucleotide exchange reaction. Anal Bioanal Chem 406, 4147–4156 (2014). https://doi.org/10.1007/s00216-014-7795-7

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  • DOI: https://doi.org/10.1007/s00216-014-7795-7

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