The guanine nucleotide-binding protein Ras exists in solution in two different conformational states when complexed with different GTP analogs such as GppNHp or GppCH 2 p. State 1 has only a very low affinity to effectors and seems to be recognized by guanine nucleotide exchange factors, whereas state 2 represents the high affinity effector binding state. In this work we investigate Ras in complex with the physiological nucleoside triphosphate GTP. By polarization transfer 31 P NMR experiments and effector binding studies we show that Ras(wt)⅐Mg 2؉ ⅐GTP also exists in a dynamical equilibrium between the weakly populated conformational state 1 and the dominant state 2. At 278 K the equilibrium constant between state 1 and state 2 of C-terminal truncated wild-type Ras(1-166) K 12 is 11.3. K 12 of full-length Ras is >20, suggesting that the C terminus may also have a regulatory effect on the conformational equilibrium. The exchange rate (k ex ) for Ras(wt)⅐Mg 2؉ ⅐GTP is 7 s ؊1 and thus 18-fold lower compared with that found for the Ras⅐GppNHp complex. The intrinsic GTPase activity substantially increases after effector binding for the switch I mutants Ras(Y32F), (Y32R), (Y32W), (Y32C/ C118S), (T35S), and the switch II mutant Ras(G60A) by stabilizing state 2, with the largest effect on Ras(Y32R) with a 13-fold increase compared with wild-type. In contrast, no acceleration was observed in Ras(T35A). Thus Ras in conformational state 2 has a higher affinity to effectors as well as a higher GTPase activity. These observations can be used to explain why many mutants have a low GTPase activity but are not oncogenic.The guanine nucleotide-binding protein Ras is involved in cellular signal transduction pathways inducing proliferation, differentiation, or apoptosis of cells. It functions as a molecular switch, cycling between an inactive GDP-bound state and an active GTP-bound state. In the active conformation Ras is able to bind different effector proteins such as Raf kinase or RalGDS with nanomolar affinity by interacting with its switch I region. Thus signals can be transmitted resulting in the corresponding cellular response.When bound to guanosine triphosphates Ras exists in two different conformational states, defined as states 1 and 2, which are in chemical equilibrium in solution (1-3). These conformational states are actually only defined for Ras with guanosine triphosphate bound (T), a state that may be different from a state with guanine diphosphate bound (D). Therefore, we will denote the two states in the following as state 1(T) and 2(T) whenever the nucleotide ligand is of concern. The equilibrium between the two states is strongly influenced by the nature of the guanine nucleotide bound to Ras and can be shifted by interaction with effector proteins or regulators such as GTPase activation proteins (GAPs).2 It was found that GTP analogs GppNHp and GppCH 2 p partially shift the equilibrium toward state 1. For the complex of wild-type Ras with physiological GTP itself the existence of the two states could not be...