Assessing the Influence of Mutation on GTPase Transition States by Using X-ray Crystallography, 19 F NMR, and DFT Approaches

Angew Chem Int Ed Engl. 2017 Aug 7;56(33):9732-9735. doi: 10.1002/anie.201703074. Epub 2017 May 24.


We report X-ray crystallographic and 19 F NMR studies of the G-protein RhoA complexed with MgF3- , GDP, and RhoGAP, which has the mutation Arg85'Ala. When combined with DFT calculations, these data permit the identification of changes in transition state (TS) properties. The X-ray data show how Tyr34 maintains solvent exclusion and the core H-bond network in the active site by relocating to replace the missing Arg85' sidechain. The 19 F NMR data show deshielding effects that indicate the main function of Arg85' is electronic polarization of the transferring phosphoryl group, primarily mediated by H-bonding to O3G and thence to PG . DFT calculations identify electron-density redistribution and pinpoint why the TS for guanosine 5'-triphosphate (GTP) hydrolysis is higher in energy when RhoA is complexed with RhoGAPArg85'Ala relative to wild-type (WT) RhoGAP. This study demonstrates that 19 F NMR measurements, in combination with X-ray crystallography and DFT calculations, can reliably dissect the response of small GTPases to site-specific modifications.

Keywords: 19F NMR; DFT calculations; GTPases; RhoA/RhoGAP; enzyme mechanisms.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Crystallography, X-Ray
  • Density Functional Theory*
  • Fluorine / chemistry
  • GTP Phosphohydrolases / chemistry
  • GTP Phosphohydrolases / genetics*
  • GTP Phosphohydrolases / metabolism
  • Magnetic Resonance Spectroscopy
  • Models, Molecular
  • Mutation


  • Fluorine
  • GTP Phosphohydrolases