HIGH-FREQUENCY (139.5 GHZ) ELECTRON-PARAMAGNETIC-RESONANCE CHARACTERIZATION OF MN(II)-(H2O)-O-17 INTERACTIONS IN GDP AND GTP FORMS OF P21 RAS

As a molecular switch, the ras protein p21 undergoes structural change s that couple recognition sites on the protein surface to the guanine nucleotide-divalent metal ion binding site, X-ray crystallographic stu dies of p21 suggest that coordination between threonine-35 and the div alent metal ion plays an important role in these conformational change s, Recent ESEEM studies of p21 in solution, however, place threonine-3 5 more distant ii om the metal and were interpreted as weak or indirec t coordination of this residue, We report high frequency (139.5 GHz) E PR spectroscopy of p21 . Mn(II) complexes of two guanine nucleotides t hat probes the link between threonine-35 and the divalent metal ion, B y analysis of high-frequency EPR spectra, we determine the number of w ater molecules in the first coordination sphere of the manganous ion t o be four in p21 . Mn(II). GDP, consistent with prior low-frequency EP R and X-ray crystallographic studies, In the complex of p21 with a GTP analog, p21 . Mn(II). GMPPNP, we determine the hydration number to be 2, also consistent with crystal structures. This result rules out ind irect coordination of threonine-35 in the solution structure of p21 . Mn(II). GMPPNP, and implicates direct, weak coordination of this resid ue as suggested by Halkides et al. [(1994) Biochemistry 33, 4019]. The O-17 hyperfine coupling constant of (H2O)-O-17 is determined as 0.25 mT in the GDP Corm and 0.28 mT in the GTP form, These values are simil ar to reported values for O-17-enriched aquo ligands and some phosphat o ligands in Mn(II) complexes. The high magnetic field strength (4.9 T ) employed in these 139.5 GHz EPR measurements leads to a narrowing of the Mn(II) EPR lines that facilitates the determination of O-17 hyper fine interactions.