Zusammenfassung
The guanine nucleotide-binding protein Ras occurs in solution in two different conformational states, state 1 and state 2 with an equilibrium constant K(12) of 2.0, when the GTP analogue guanosine-5'-(beta,gamma-imido)triphosphate or guanosine-5'-(beta,gamma-methyleno)triphosphate is bound to the active centre. State 2 is assumed to represent a strong binding state for effectors with a ...
Zusammenfassung
The guanine nucleotide-binding protein Ras occurs in solution in two different conformational states, state 1 and state 2 with an equilibrium constant K(12) of 2.0, when the GTP analogue guanosine-5'-(beta,gamma-imido)triphosphate or guanosine-5'-(beta,gamma-methyleno)triphosphate is bound to the active centre. State 2 is assumed to represent a strong binding state for effectors with a conformation similar to that found for Ras complexed to effectors. In the other state (state 1), the switch regions of Ras are most probably dynamically disordered. Ras variants that exist predominantly in state 1 show a drastically reduced affinity to effectors. In contrast, Ras(wt) bound to the GTP analogue guanosine-5'-O-(3-thiotriphosphate) (GTPgammaS) leads to (31)P NMR spectra that indicate the prevalence of only one conformational state with K(12) > 10. Titration with the Ras-binding domain of Raf-kinase (Raf-RBD) shows that this state corresponds to effector binding state 2. In the GTPgammaS complex of the effector loop mutants Ras(T35S) and Ras(T35A) two conformational states different to state 2 are detected, which interconvert over a millisecond time scale. Binding studies with Raf-RBD suggest that both mutants exist mainly in low-affinity states 1a and 1b. From line-shape analysis of the spectra measured at various temperatures an activation energy DeltaH(|) (1a1b) of 61 kJ.mol(-1) and an activation entropy DeltaS(|) (1a1b) of 65 J.K(-1).mol(-1) are derived. Isothermal titration calorimetry on Ras bound to the different GTP-analogues shows that the effective affinity K(A) for the Raf-RBD to Ras(T35S) is reduced by a factor of about 20 compared to the wild-type with the strongest reduction observed for the GTPgammaS complex.
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