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Assessing the influence of mutation on GTPase transition states using x-ray crystallography, 19F NMR, and DFT approaches

Waltho, Jonathan P, Jin, Yi, Molt Jr., Robert W., Pellegrini, Erika, Cliff, Matthew J, Bowler, Matthew W., Richards, Nigel G. J. and Blackburn, G. Michael 2017. Assessing the influence of mutation on GTPase transition states using x-ray crystallography, 19F NMR, and DFT approaches. Angewandte Chemie - International Edition 56 (33) , pp. 9732-9735. 10.1002/anie.201703074

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Abstract

We report X-ray crystallographic and 19F NMR studies of RhoA complexed with MgF3-, GDP, and RhoGAP having the mutation Arg85'Ala, which, when combined with DFT calculations, permit the identification of changes in TS properties. Thus, X-ray shows how Tyr34 maintains solvent exclusion and the core H-bond network in the active site by relocating to replace the missing Arg85' sidechain. 19F 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 GTP hydrolysis is higher in energy when RhoA is complexed with RhoGAPArg85'Ala relative to WT RhoGAP. This study demonstrates that 19F NMR measurements, in combination with X-ray and DFT, can reliably dissect the response of small GTPases to site-specific modification.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Chemistry
Subjects: Q Science > QD Chemistry
Publisher: Wiley-Blackwell
ISSN: 1433-7851
Funders: BBSRC
Date of First Compliant Deposit: 14 June 2017
Date of Acceptance: 12 May 2017
Last Modified: 25 Jul 2019 16:09
URI: http://orca.cf.ac.uk/id/eprint/100642

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