THE HYDRATION OF RAS P21 IN SOLUTION DURING GTP HYDROLYSIS BASED ON SOLUTION X-RAY-SCATTERING PROFILE

The small-angle X-ray scattering technique was used to characterize the structure in solution of wild type ras p21 as well as the oncogenic proteins mutated at residue 12, 59, or 61. In the presence of GDP, the radius of gyration, R,, determined for wild type ras p21 was 16.89+/-0.01 Angstrom, while the wild type ras p21 bound to the GTP analogue GDPNHP (5'-guanyl imido diphosphate beta-gamma-imidoguanosine 5'-triphosphate) showed an R(g) value of 17.46+/-0.01 Angstrom, which is 3.3% larger. The result shows that ras p21 expands upon GTP binding. The R(g)s of mutated proteins were 17.04+/-0.01, 16.98+/-0.01, and 17.03+/-0.01 Angstrom for the Gly-12 to Val, Ala-59 to Thr, and Gln-61 to Leu mutants, respectively. The scattering profiles were analyzed by simulation of hydrated ras p21, based on the crystal atomic coordinates, and it was concluded that the ras p21 molecule incorporates 20% more bulk water upon GTP binding. The increase of bulk water is especially conspicuous around the interface between switch I (residues 32-40) and switch II (residues 60-66) regions. This suggests that hydration plays an important role in the interaction with GAP.