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

Christopher J. Halkides, Brendan F. Bellew, Gary J. Gerfen, Christian T. Farrar, Percy H. Carter, Bernice Ruo, David A. Evans, Robert G. Griffin, David J. Singel

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)12194-12200
Number of pages7
JournalBiochemistry
Volume35
Issue number37
DOIs
Publication statusPublished - Sep 30 1996
Externally publishedYes

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ASJC Scopus subject areas

  • Biochemistry

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