HIGH-FREQUENCY (139.5 GHZ) ELECTRON-PARAMAGNETIC-RESONANCE SPECTROSCOPY OF THE GTP FORM OF P21 RAS WITH SELECTIVE O-17 LABELING OF THREONINE

Electron paramagnetic resonance spectroscopy at 139.5 GHz has been use d to study p21 I-ns complexed with Mn(II) and guanosine 5'-(beta,gamma -imidotriphosphate), an analog of GTP. The p21 sample studied was sele ctively labeled with [O-17(gamma)]threonine to a final enrichment of 3 0%, A Mn(II)-O-17 hyperfine interaction was observed, but the value of the coupling constant, 0.11 +/- 0.04 mT, is the smallest such value y et reported, Ab initio calculations indicate that this value is consis tent with direct coordination of the threonine hydroxyl group and prov ide an estimate for the Mn(II)-O-17 bond length of 2.7 Angstrom. The m easured hyperfine coupling constant and associated bond length starkly contrast with typical values for Mn(II)-O-17 coordination complexes, namely similar to 0.25 mT and similar to 2.2 Angstrom, respectively. T his contrast underscores the peculiar weakness of this Mn(II)-O intera ction in p21 and persuasively argues that the nucleotide-induced confo rmational change, which is known to encompass the region of p21 involv ing Thr35, is not driven by Mn(II) coordination of the Thr35 hydroxyl group.