Structure of the metal-water complex in Ras x GDP studied by high-field EPR spectroscopy and 31P NMR spectroscopy.
The small GTPase Ras plays a key role as a molecular switch in the intercellular signal transduction. On Mg(2+) --> Mn(2+) substituted samples, the first ligand sphere of the metal ion in the inactive, GDP-bound Ras has been studied by continuous wave EPR at 94 GHz (W-band). Via replacement of normal water with (17)O-enriched water, the (17)O--(55)Mn superhyperfine coupling was used to determine the number of water ligands bound to the metal ion. In contrast 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 same 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 mutant Ras(G12V), four water ligands can be observed in liquid solution. The EPR studies were supplemented by (31)P NMR studies on the Mg(2+) x GDP complexes of the wild-type protein and the three mutants. Ras(T35A) exists in two conformational states (1 and 2) with an equilibrium constant K(1)(1,2) of approximately 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 observed 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.
Rohrer
M
M
Institute for Physical and Theoretical Chemistry, University of Frankfurt, Frankfurt, Germany.
Prisner
T F
TF
Brügmann
O
O
Käss
H
H
Spoerner
M
M
Wittinghofer
A
A
Kalbitzer
H R
HR
D019943:Amino Acid Substitution / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D006153:Guanosine Diphosphate / Q000737:chemistry*; D046911:Macromolecular Substances; D008345:Manganese / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D017354:Point Mutation; D012996:Solutions; D013696:Temperature; D014867:Water / Q000737:chemistry*; D018631:ras Proteins / Q000737:chemistry* / Q000235:genetics
D046911:Macromolecular Substances; D012996:Solutions; D014867:Water; D006153:Guanosine Diphosphate; D008345:Manganese; D018631:ras Proteins