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Measuring G-protein-coupled Receptor Signaling via Radio-labeled GTP Binding
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The protonation states of GTP and GppNHp in Ras proteins

Daniel Mann1, Jörn Güldenhaupt1, Jonas Schartner1

  • 1From the Department of Biophysics, Ruhr University Bochum, 44780 Bochum, Germany and.

The Journal of Biological Chemistry
|February 1, 2018
PubMed

Insights

Ras signaling is crucial for cell proliferation and cancer. This study reveals guanosine triphosphate (GTP) is fully deprotonated in Ras

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Structural Biology

Background:

  • The small GTPase Ras is a key regulator of cellular signaling pathways, particularly cell proliferation.
  • Ras function is intrinsically linked to guanosine triphosphate (GTP) hydrolysis, a process implicated in diseases like cancer.
  • Previous studies on Ras mechanism have overlooked the potential protonation state of GTP.

Purpose of the Study:

  • To elucidate the detailed prehydrolysis state of Ras-bound GTP.
  • To investigate the protonation state of the guanosine triphosphate (GTP) group in Ras.
  • To reconcile conflicting findings regarding GTP protonation in Ras signaling.

Main Methods:

  • Quantum mechanics/molecular mechanics (QM/MM) simulations to calculate infrared and NMR spectra.
  • Comparison of simulated spectra with existing experimental data.
  • Experimental measurement of infrared spectra for GTP and analogs bound to lipidated Ras on a membrane system under near-native conditions.

Main Results:

  • Simulated infrared and NMR spectra were computed to analyze the Ras prehydrolysis state.
  • Experimental infrared spectra were obtained for Ras-bound GTP and analogs on a membrane.
  • Findings support a structural model where guanosine triphosphate (GTP) is fully deprotonated in the Ras prehydrolysis state.

Conclusions:

  • The study provides a unified model for the Ras prehydrolysis state.
  • Evidence confirms that guanosine triphosphate (GTP) is fully deprotonated when bound to Ras before hydrolysis.
  • This finding resolves previous questions about GTP protonation in Ras signaling.
Keywords:
Fourier transform IR, FTIRGTP hydrolysisGTPaseNMRQM/MM simulationRas proteinmolecular dynamicsnuclear magnetic resonancequantum chemistry

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