UCL Discovery
UCL home » Library Services » Electronic resources » UCL Discovery

Mechanism of Nucleotide Exchange by Guanine Nucleotide Exchange Factors on the Ras Superfamily of Small G-Proteins

Pickford, Christopher; (2000) Mechanism of Nucleotide Exchange by Guanine Nucleotide Exchange Factors on the Ras Superfamily of Small G-Proteins. Doctoral thesis (Ph.D), UCL (University College London). Green open access

[thumbnail of Mechanism_of_nucleotide_exchan.pdf] Text
Mechanism_of_nucleotide_exchan.pdf

Download (15MB)

Abstract

The Ras superfamily of small G-proteins cycle between an active GTP bound form and an inactive GDP bound form. This cycle is controlled by GTPase activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs). Sos, a GEF for Ras proteins, is a ubiquitously expressed 150 kDa protein that is recruited to the cell membrane by Grb2 on phosphorylation of a receptor, where it promotes GDP release from Ras, enabling formation of the active Ras.GTP complex. Study of the interaction of the catalytic domain of mSos1 (CmSos1) with N-Ras in vitro was hampered by low expression levels of CmSos1 in E.coli. The gene for CmSos1, coding for residues 577-1076, was reconstructed following codon optimisation and expressed in E.coli. Yields for a 4 Litre culture were 120 mg of pure CmSos1 protein, an ~100 fold enhancement in expression levels. This enabled the interaction of CmSos1 with N-Ras to be studied, using the fluorescent analogue of GDP, 2-deoxy-3-O-N-methlyanthraniloylGDP (mdGDP) for transient kinetic and equilibrium measurements. The mechanism of nucleotide exchange has been proposed to be a substituted mechanism, exchange factors stabilising a nucleotide free binary complex of GEF.G- protein allowing rebinding of nucleotide via a transient ternary complex. The formation of a weak ternary N-Ras.mdGDP.CmSos1 complex has been studied using fluorescence anisotropy measurements. Addition of excess GDP to N- Ras.mdGDP.CmSos1 enabled the release of mdGDP from the ternary complex to be analysed. These processes have been followed over millisecond time scales using stopped-flow apparatus. The nucleotide free N-Ras.CmSos1 complex has been purified and characterised. The reverse reaction, analysing the association of nucleotide to the N-Ras.CmSos1 complex, has been studied. Computer simulation of this data has led to rate and equilibrium constants for the steps of the reaction being proposed. The interaction of different nucleotides and nucleotide bound states has also been observed. The interaction of the proposed catalytic domain of aPIX (a Dbl homology exchange factor) with Rac1 has been studied. Low exchange activity has been observed, leading to the proposal that other factors are required for the observed in vivo activity of PIX on Rho family proteins.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Mechanism of Nucleotide Exchange by Guanine Nucleotide Exchange Factors on the Ras Superfamily of Small G-Proteins
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Pure sciences; Ras superfamily
URI: https://discovery.ucl.ac.uk/id/eprint/10097881
Downloads since deposit
51Downloads
Download activity - last month
Download activity - last 12 months
Downloads by country - last 12 months

Archive Staff Only

View Item View Item