Vibrational structure of GDP and GTP bound to RAS: An isotope-edited FTIR study

A complete vibrational description of the bonding of a ligand to a protein requires the assignment of both symmetric and antisymmetric vibrational mod es. The symmetric modes of isotopically enriched enzyme-bound ligands can b e obtained by Raman difference spectroscopy, but until now, the antisymmetr ic modes, which require IR difference spectroscopy, have not been generally accessible. We have developed the methodology needed to perform IR differe nce spectroscopy, assign the antisymmetric modes, and accurately describe b onding. The method is used to assess the bonding changes that occur as Mg G DP and Mg GTP move from solution into the active site of RAS. Binding to RA S opens the nonbridging, O chemical anion P chemical anion O angle of the g amma -phosphate of GTP by 2.7 degrees, yet the angular freedom (dispersion of the O chemical anion P chemical anion O angle) of the gamma -phosphate i s comparable to that in solution. In contrast, the motion of the beta -phos phate of GDP is highly restricted, suggesting that it positions the gamma - phosphate for nucleophilic attack. The beta,gamma -bridging O-P bond of bou nd GTP is slightly weakened, being lengthened by 0.005 Angstrom in the acti ve site, corresponding to a bond order decrease of 0.012 valence unit (vu). The observed binding changes are consistent with a RAS-mediated hydrolysis mechanism that parallels that for solution hydrolysis.