Bf. Bellew et al., HIGH-FREQUENCY (139.5 GHZ) ELECTRON-PARAMAGNETIC-RESONANCE CHARACTERIZATION OF MN(II)-(H2O)-O-17 INTERACTIONS IN GDP AND GTP FORMS OF P21 RAS, Biochemistry, 35(37), 1996, pp. 12186-12193
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.