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Abstract

31P NMR revealed that the complex of p21ras with the GTP analog GppNHp·Mg2+ exists in two conformational states, states 1 and 2. In wild-type p21ras the equilibrium constant between the two states is 1.09. The population of these states is different for various mutants but independent of temperature. The activation enthalpy H and activation entropy S for the conformational transitions were determined by full-exchange matrix analysis for wild-type p21ras and p21ras(S65P). For the wild-type protein one obtains H = 89 ± 2 kJ mol-1 and S = 102 ± 20 J mol-1 K-1 and for the mutant protein H = 93 ± 7 kJ mol-1 and S = 138 ± 30 J mol-1 K-1. The study of various p21ras mutants suggests that the two states correspond to different conformations of loop L2, with Tyr-32 in two different positions relative to the bound nucleotide. High-field EPR at 95 GHz suggests that the observed conformational transition does not directly influence the coordination sphere of the protein-bound metal ion. The influence of this transition on loop L4 was studied by 1H NMR with mutants E62H and E63H. There was no indication that L4 takes part in the transition described in L2, although a reversible conformational change could be induced by decreasing the pH value. The exchange between the two states is slow on the NMR time scale (<10 s-1); at approximately pH 5 the population of the two states is equal. The interaction of p21ras-triphosphate complexes with the Ras-binding domain (RBD) of the effector protein c-Raf-1, Raf-RBD, and with the GTPase activating protein GAP was studied by 31P NMR spectroscopy. In complex with Raf-RBD the second conformation of p21ras (state 2) is stabilized. In this conformation Tyr-32 is located in close proximity to the phosphate groups of the nucleotide, and the -phosphate resonance is shifted upfield by 0.7 ppm. Spectra obtained in the presence of GAP suggest that in the ground state GAP does not interact directly with the nucleotide bound to p21ras and does not induce larger conformational changes in the neighborhood of the nucleotide. The experimental data are consistent with a picture where GAP accelerates the exchange process between the two states and simultaneously increases the population of state 1 at higher temperature