Characterization of the active site of p21 ras by electron spin-echo envelope modulation spectroscopy with selective labeling : comparisons between GDP and GTP forms

Selectively labeled samples of human H- or N-ras p21 ligated to MnIIGDP or MnIIGMPPNP were studied by electron spin-echo envelope modulation spectroscopy in order to define the protein environment around the divalent metal. We incorporated (4-13C)-labeled Asx into p21-MnI1GDP and found that the distance from the carboxyl 13C of Asp57 to MnIIis -4.1 A. Our result is consistent with indirect coordination of this residue to the metal. From a (2-ZH)Thr-labeled sample, we estimate that the distance from the Mn" ion to the ZH of Thr35 is at least 5.8 A. Thus, the only protein or nucleotide ligands to the metal appear to be Serl7 and the p a (4-13C)-labeled Asx sample of p21 gives a distance of -4 A between Mn" and the label of Asp57, again implying indirect coordination. Both of these values are very similar to those found for the GDP form of the protein. The results for Thr35, however, reveal a structural difference between the GDP and GTP forms in the region of Thr35. In addition, the position of this residue is found to be different from the crystal structure and in a manner suggesting that the metal ligation of Thr35 does not drive the conformational change that accompanies nucleotide substitution.