TY - JOUR
T1 - Vibrational structure of GDP and GTP bound to RAS
T2 - An isotope-edited FTIR study
AU - Cheng, H.
AU - Sukal, S.
AU - Deng, H.
AU - Leyh, T. S.
AU - Callender, R.
PY - 2001/4/3
Y1 - 2001/4/3
N2 - A complete vibrational description of the bonding of a ligand to a protein requires the assignment of both symmetric and antisymmetric vibrational modes. The symmetric modes of isotopically enriched enzyme-bound ligands can be obtained by Raman difference spectroscopy, but until now, the antisymmetric modes, which require IR difference spectroscopy, have not been generally accessible. We have developed the methodology needed to perform IR difference spectroscopy, assign the antisymmetric modes, and accurately describe bonding. The method is used to assess the bonding changes that occur as Mg·GDP and Mg·GTP move from solution into the active site of RAS. Binding to RAS opens the nonbridging, O-P-O angle of the γ-phosphate of GTP by 2.7°, yet the angular freedom (dispersion of the O-P-O angle) of the γ-phosphate is comparable to that in solution. In contrast, the motion of the β-phosphate of GDP is highly restricted, suggesting that it positions the γ-phosphate for nucleophilic attack. The β,γbridging O-P bond of bound GTP is slightly weakened, being lengthened by 0.005 Å in the active 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.
AB - A complete vibrational description of the bonding of a ligand to a protein requires the assignment of both symmetric and antisymmetric vibrational modes. The symmetric modes of isotopically enriched enzyme-bound ligands can be obtained by Raman difference spectroscopy, but until now, the antisymmetric modes, which require IR difference spectroscopy, have not been generally accessible. We have developed the methodology needed to perform IR difference spectroscopy, assign the antisymmetric modes, and accurately describe bonding. The method is used to assess the bonding changes that occur as Mg·GDP and Mg·GTP move from solution into the active site of RAS. Binding to RAS opens the nonbridging, O-P-O angle of the γ-phosphate of GTP by 2.7°, yet the angular freedom (dispersion of the O-P-O angle) of the γ-phosphate is comparable to that in solution. In contrast, the motion of the β-phosphate of GDP is highly restricted, suggesting that it positions the γ-phosphate for nucleophilic attack. The β,γbridging O-P bond of bound GTP is slightly weakened, being lengthened by 0.005 Å in the active 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.
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U2 - 10.1021/bi0021131
DO - 10.1021/bi0021131
M3 - Article
C2 - 11300784
AN - SCOPUS:0035799325
SN - 0006-2960
VL - 40
SP - 4035
EP - 4043
JO - Biochemistry
JF - Biochemistry
IS - 13
ER -