Nucleotide hydrolysis-dependent conformational changes in p21(ras) as studied using ESR spectroscopy

Abstract

We have employed ESR spectroscopy using guanine nucleotides that contain a spin label at the 2',3'-position of the ribose to investigate structural changes in the proto-oncogene product p21(ras) that are dependent on nucleotide hydrolysis. The three nucleotide analogs used were 2',3'-(2,2,5,5- tetramethyl-3-pyrroline-1-oxyl-3carboxylic acid ester (SL) GTP, SL-GDP, and the nonhydrolyzable analog SL-guanylylimidodiphosphate. SL-GTP was hydrolyzed by p21 with rates similar to those for GTP hydrolysis and appears to be an excellent substrate analog. The ESR spectra of SL-GTP and SL-GDP in complex with p21 differ significantly when acquired at 0 °C or 5 °C indicating different environments (conformations) of the protein-bound radicals depending on the phosphorylation state of the bound nucleotide. We calculated the rate constant for the conformational change as deduced from the changes in the corresponding ESR spectra upon incubation of the p21·SL-GTP complex at 25 °C and compared it to the rate constant of hydrolysis of SL-GTP at the same temperature. The rate constant deduced from the ESR method was similar to that determined by a high performance liquid chromatography technique. The data are in agreement with the idea that a conformational change during GTP hydrolysis by p21 occurs simultaneously with the actual hydrolysis step.