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 th e 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 G DPNHP (5'-guanyl imido diphosphate beta-gamma-imidoguanosine 5'-tripho sphate) showed an R(g) value of 17.46+/-0.01 Angstrom, which is 3.3% l arger. 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 mu tants, respectively. The scattering profiles were analyzed by simulati on of hydrated ras p21, based on the crystal atomic coordinates, and i t was concluded that the ras p21 molecule incorporates 20% more bulk w ater upon GTP binding. The increase of bulk water is especially conspi cuous around the interface between switch I (residues 32-40) and switc h II (residues 60-66) regions. This suggests that hydration plays an i mportant role in the interaction with GAP.