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.

Label-Free Time-Gated Luminescent Detection Method for the Nucleotides with Varying Phosphate Content

“…1198-1530) were of human origin and produced in E. coli [17][18][19]. Eu(III)-GTP was synthetized and conjugated as described elsewhere [18][19][20]. The soluble quencher molecule, MT2, was obtained from QRET Technologies (Turku, Finland), and used according to manufacturer's instruction.…”

“…The Eu(III)-GTP association assay was used as a control assay for label-free GTP association detection using Probe 1 [18]. Eu(III)-GTP association assay was performed in 10 µL volume using 200 nM K-Ras (wild-type, and mutants G12C, G12D, Q61L, and Q61R), SOS cat (200 nM), Eu(III)-GTP (10 nM), and MT2 (1.5 µM).…”

“…We have previously described antibody-based nucleotide detection system for GTP and cAMP using single-label technique called quenching resonance energy transfer (QRET) [15][16][17][18][19]. However, nucleotide detection using antibodies is limited to affinity of the antibody and detection of multiple different nucleotides requires the usage of different antibodies.…”

Quenching resonance energy transfer (QRET): a single-label technique for inhibitor screening and interaction studies

“…Like the Transcreener FP assay, QRET enables small GTPase inhibitor screening in singlelabel fashion (Fig. 2b) [51,52]. The QRET technique is based on Eu 3+ -GTP monitoring, unlike the Transcreener method which monitors GDP produced after GTP hydrolysis.…”

“…GTP monitoring in QRET is advantageous over GDP, because it can enable an easy detection of both GTP association (small GTPase activation) and GTP hydrolysis (small GTPase inactivation) [53]. In addition, QRET for GTP association detection has functional elements, such as the association reaction which can be monitored kinetically in a microtiter plate format [52]. Both the QRET assay for GTP association and GTP hydrolysis detection have been shown to be feasible in 1280 small compound pilot screens (Kopra et al, unpublished data).…”