High frequency (139.5 GHz) electron paramagnetic resonance spectroscopy of the GTP form of p21 ras with selective ¹⁷O labeling of threonine

Electron paramagnetic resonance spectroscopy at 139.5 GHz has been used to study p21 ras complexed with Mn(II) and guanosine 5'-(β,γ- imidotriphosphate), an analog of GTP. The p21 sample studied was selectively labeled with [¹⁷O(γ)]threonine to a final enrichment of 30%. A Mn(II)- ¹⁷O 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)-¹⁷O bond length of 2.7 A. The measured hyperfine coupling constant and associated bound length starkly contrast with typical values for Mn(II)-¹⁷O coordination complexes, namely, ~0.25 mT and ~2.2 Å, 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.