Ludbrook, Steven Bryan; (1997) Biochemical characterisation of rho/rac GTPases and their GTPase activating proteins in humans and Dictyostelium discoideum. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This project falls into two parts. Firstly to investigate the mechanism of the interaction of the human small GTPase rhoA with the GTPase activating protein rhoGAP. Secondly to study the functional roles of rho subfamily GTPases in D. discoideum and to identify GTPase activating proteins (GAPs) for these proteins. For the first part, rho A and rhoGAP catalytic domain proteins have been purified from over-expressing cultures of E. coli as recombinant proteins. The elementary kinetic constants in the GTPase cycle of rhoA have been determined biochemically both using native guanine nucleotides and fluorescent guanine nucleotide analogues, which are 2'(3')-O-(N-methylanthraniloyl) (mant) derivatives of GTP, GDP, and GMPPNP. The interaction of rhoA with rhoGAP was studied by stopped-flow and quenched-flow techniques. The binding of rhoA.mantGMPPNP with rhoGAP is proposed to occur as a two-step process, having an initial rapid equilibrium process with a KD of 11.9 μM at 20°C in 20mM Tris-HCl pH7.5, 2mM MgCl2, ImM DTT, followed by an isomerisation of the rhoA.mantGMPPNP.rhoGAP complex having a rate constant of 440 s-1. The binding of rhoA.mantGTP to rhoGAP was also shown to be a two-step process under the same conditions, with an initial rapid equilibrium process with a KD of 8.7 μM, followed by an isomerisation of the rhoA.mantGTP.rhoGAP complex having a rate constant of 29.7 s-1. The isomerisation of the rhoA.mantGTP.rhoGAP complex is followed by cleavage of the rho-bound mantGTP with a rate constant of 6.0 s-1. This cleavage process is followed by dissociation of rhoGAP with a rate constant of 1.36 s-1. For the second part, previously described rho subfamily genes have been cloned from D.discoideum and specific activated and dominant negative mutants made. The proteins have been purified from over-expressing cultures of E.coli as recombinant proteins and the elementary kinetic constants in the GTPase cycle have been determined by biochemical techniques. The characterised raclA and racC purified proteins have been used to detect GAP activity in D. discoideum cytosol by biochemical assays, with at least five peaks of GAP activity of different substrate specificity identified from an anion- exchange purification. These assays for GAP activity have been complemented by a direct cloning approach using PCR to identify rhoGAP homologues in D. discoideum, which resulted in the cloning of a racGAP, called DdracGAP. DdracGAP is a large (~150kDa) protein with SH3, DH, and PH domains in addition to the GAP domain. The GAP domain has been shown to be functionally active towards rac1A, racC, and human rhoA and racl, but not human N-ras. Finally structural investigations have been performed on many of the proteins described in this thesis in collaboration with the Division of Protein Structure at N.I.M.R.. The results of this are the structures of human rhoGAP catalytic domain to 2.0 Angstrom resolution and D.discoideum raclA.GMPPNP to 3.0 Angstrom resolution.

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