Structure of the metal-water complex in Ras center dot GDP studied by high-field EPR spectroscopy and P-31 NMR spectroscopy

The small GTPase Ras plays a key role as a molecular switch in the intercel lular signal transduction. On Mg2+ -> Mn2+ substituted samples, the first l igand sphere of the metal ion in the inactive, GDP-bound Ras has been studi ed by continuous wave EPR at 94 GHz (W-band). Via replacement of normal wat er with O-17-enriched water, the (O-55Mn)-O-17 superhyperfine coupling was used to determine the number of water ligands bound to the metal ion. In co ntrast to EPR data on frozen solutions and X-ray data from single crystals where four direct ligands to the metal ion are found, the wild-type protein has only three water ligands bound in solution at room temperature. The sa me number of water ligands is found for the mutant Ras(T35S). However, for the alanine mutant in position 35 Ras(T35A) as well as for the oncogenic mu tant Ras(G12V), four water ligands can be observed in liquid solution. The EPR studies were supplemented by P-31 NMR studies on the Mg2+ GDP complexes of the wild-type protein and the three mutants. Ras(T35A) exists in two co nformational states (1 and 2) with an equilibrium constant K-1(1,2) of appr oximately 0.49 and rate constants k(1-1) which are much smaller than 40 s(- 1) at 298 K. For wild-type Ras and Ras(T35S), the two states can also be ob served with equilibrium constants K-1(1,2) of approximately 0.31 and 0.21, respectively. In Ras(G12V), only one conformational state could be detected .