High Frequency (139.5 GHz) Electron Paramagnetic Resonance Spectroscopy of the GTP Form of p21 ras with Selective 17O Labeling of Threonine

• Published in: Biochemistry (Easton) Vol. 35; no. 37; pp. 12194 - 12200
• Main Authors: Halkides, Christopher J, Bellew, Brendan F, Gerfen, Gary J, Farrar, Christian T, Carter, Percy H, Ruo, Bernice, Evans, David A, Griffin, Robert G, Singel, David J
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 17.09.1996
• Subjects: Binding Sites; Electron Spin Resonance Spectroscopy - methods; Gas Chromatography-Mass Spectrometry; Guanosine Triphosphate - metabolism; Guanylyl Imidodiphosphate - metabolism; Kinetics; Manganese - metabolism; Oxygen Isotopes; Protein Binding; Protein Conformation; Proto-Oncogene Proteins p21(ras) - chemistry; Proto-Oncogene Proteins p21(ras) - metabolism; Threonine
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi9605954

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 [17Oγ]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 Å. The measured hyperfine coupling constant and associated bond 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.