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 used to study p21 I-ns complexed with Mn(II) and guanosine 5'-(beta,gamma-imidotriphosphate), an analog of GTP. The p21 sample studied was selectively labeled with [O-17(gamma)]threonine to a final enrichment of 30%, 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 yet reported, Ab initio calculations indicate that this value is consistent with direct coordination of the threonine hydroxyl group and provide an estimate for the Mn(II)-O-17 bond length of 2.7 Angstrom. The measured 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. This contrast underscores the peculiar weakness of this Mn(II)-O interaction in p21 and persuasively argues that the nucleotide-induced conformational change, which is known to encompass the region of p21 involving Thr35, is not driven by Mn(II) coordination of the Thr35 hydroxyl group.