title: High frequency (139.5 GHz) electron paramagnetic resonance spectroscopy of the GTP form of p21 ras with selective 17O labeling of threonine.
abstract: Electron paramagnetic resonance spectroscopy at 139.5 GHz has been used to study p21 ras complexed with Mn(II) and guanosine 5'-(beta, gamma-imidotriphosphate), an analog of GTP. The p21 sample studied was selectively labeled with [17O gamma]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 bond length starkly contrast with typical values for Mn(II)-17O coordination complexes, namely, approximately 0.25 mT and approximately 2.2 A, 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.
authors.affiliation: Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02154, USA.
authors.forename: C J
authors.initials: CJ
authors.lastname: Halkides
authors.forename: B F
authors.initials: BF
authors.lastname: Bellew
authors.forename: G J
authors.initials: GJ
authors.lastname: Gerfen
authors.forename: C T
authors.initials: CT
authors.lastname: Farrar
authors.forename: P H
authors.initials: PH
authors.lastname: Carter
authors.forename: B
authors.initials: B
authors.lastname: Ruo
authors.forename: D A
authors.initials: DA
authors.lastname: Evans
authors.forename: R G
authors.initials: RG
authors.lastname: Griffin
authors.forename: D J
authors.initials: DJ
authors.lastname: Singel
mesh_terms: D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D008401:Gas Chromatography-Mass Spectrometry; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D007700:Kinetics; D008345:Manganese / Q000378:metabolism; D010103:Oxygen Isotopes; D011485:Protein Binding; D011487:Protein Conformation; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry* / Q000378:metabolism*; D013912:Threonine
chemical_list: D010103:Oxygen Isotopes; D013912:Threonine; D006165:Guanylyl Imidodiphosphate; D008345:Manganese; D006160:Guanosine Triphosphate; D016283:Proto-Oncogene Proteins p21(ras)