Electron paramagnetic resonance measurements of the hydration of Mn(II) in ternary complexes with GDP and ras p21 proteins.
Electron paramagnetic resonance (EPR) spectroscopy has been used to determine the hydration numbers of Mn(II) in complexes with GDP and three forms of ras p21. EPR signals of Mn(II) in the GDP complex with viral-Harvey p21pRAS1 (Arg 12, Thr 59), p21EC (Gly 12, Thr 59), and p21EJ (Val 12, Thr 59) have narrow line-widths that permit ready observation of inhomogeneous broadening from unresolved superhyperfine coupling with the nuclear spin of 17O of directly coordinated oxygen ligands. Quantitative analysis of the lineshapes for the samples in H2 17O-enriched water indicates that four water ligands coordinate to the metal ion in the GDP complexes with all three proteins. The four solvent ligands, together with an oxygen from the beta-phosphate group of GDP, leave space for only one ligand from the protein. An X-ray diffraction-derived model for the MgII beta-gamma-imidoguanosine-5'-triphosphate complex with p21 shows coordination of Mg(II) to the beta- and gamma-phosphate groups of the nucleotide as well as to the hydroxyl groups of Thr 35 and Ser 17 (Pai, E.F., Kabusch, W., Krengel, U., Holmes, K. H., John, J., and Wittinghofer, A., 1989, Nature (London) 341, 209-214). Thus, upon conversion of the nucleotide from a triphosphate to a diphosphate, solvent replaces both the gamma-phosphate of the nucleotide and one of the protein ligands. The EPR results are consistent with a recent X-ray crystallographic model for the p21-MgIIGDP complex (Milburn, M. V., Tong, L., DeVos, A. M., Brunger, A., Yamaizumi, Z., Nishimura, S., and Kim, S.-H., 1990, Science 247, 939-945). EPR spectra of complexes with the three forms of ras p21 differ with respect to the intrinsic linewidths of the EPR signals. These subtle differences in linewidth appear to originate from slight differences in local disorder near the metal-nucleotide binding site.
Smithers
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GW
Institute for Enzyme Research, Graduate School, University of Wisconsin, Madison 53705.
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D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli; D006150:Guanine Nucleotides / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism*; D008345:Manganese / Q000378:metabolism*; D015689:Oncogene Protein p21(ras) / Q000378:metabolism*; D014867:Water / Q000032:analysis
D006150:Guanine Nucleotides; D014867:Water; D006153:Guanosine Diphosphate; D008345:Manganese; D015689:Oncogene Protein p21(ras)