Moore, Keith J M; (1992) Kinetic and fluorescence studies of the interaction of p21(ras) with guanine nucleotides and the GTPase activating proteins, p120-GAP and Nfi-GAP. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The protein products of the ras proto-oncogenes, p21ras, are 21 KDa guanine nucleotide binding proteins which possess a slow intrinsic GTPase activity. These proteins are thought to be involved in the regulation of cell growth and differentiation, and some single point mutations in the ras genes lead to cell transformation. The biological signal mediated by p21ras is determined by the in vivo concentration of the p21ras.GTP complex since only when bound to GTP are these proteins thought to be biologically active. Nucleotide exchange of bound GDP for cytoplasmic GTP leads to the reformation of the p21ras.GTP complex. Two proteins (GAPs) have been identified which accelerate the rate of GTP hydrolysis by wild p21ras but not by oncogenic p21ras mutants. The kinetic mechanism of the p21ras.GTPase in the presence of the catalytic-domains of two GAPs, p120-GAP and neurofibromin, has been investigated. The studies are based primarily on the use of a fluorescent GTP analogue, 2'(3')-O-(N-methylanthraniloyl)-GTP (mantGTP), which shows changes in fluorescence intensity during several elementary steps in the GTPase mechanism. The experimental results are compatible with a mechanism where the intrinsic hydrolysis of GTP by p21ras is preceded, and controlled, by a protein conformational change in the p21ras.GTP complex. p120-GAP accelerates the overall rate of GTP cleavage by promoting this rate limiting conformational change. The binding of p120-GAP to p21ras.GTP is a rapidly reversible reaction (equilibrium dissociation constant, Kd = 20 μM at l ≈ 20 mM). p120-GAP accelerates the rate of the subsequent conformational change in p21ras.GTP by a factor of 105 compared to the rate of the equivalent reaction in the absence of p120-GAP. A similar extent of activation was observed with neurofibromin although the affinity for p21N-ras.GTP was 20-fold higher than that of p120-GAP. In contrast to p120-GAP, the binding reaction with neurofibromin may not be rapidly reversible. For both proteins, increasing ionic strength lead to a marked increase in the Kd; the rate constant of the conformational change being essentially unaltered. The binding of p120-GAP and neurofibromin to the p21ras.mantGTP complex is associated with an increase in fluorescence intensity, anisotropy and energy transfer (from tryptophan residues in the GAP proteins). These fluorescence signals can be used to determine the equilibrium dissociation constants for the binding of GAP proteins to p21ras. The intrinsic GTPase activity of the Gly 12→ Pro mutant of p21ras is only weakly accelerated by either p120-GAP or neurofibromin although both GAPs bind with an affinity similar to that with the wild type p21ras protein. An explanation for the weakly transforming phenotype of this mutant compared to other Gly 12 mutants is offered. One consequence of this interpretation is that the in vivo rate of GAP-activated GTP hydrolysis and nucleotide exchange may be slower than generally assumed. Finally, the hydrodynamic properties of p21ras proteins have been investigated using time resolved fluorescence techniques combined with a range of fluorescent guanine nucleotide analogues. In all cases, the rotational correlation time of p21ras is consistent with a dimeric structure for these proteins in solution.

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