Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.796937
Title: Studies of the Escherichia coli chaperonin protein GroEL (cpn 60)
Author: Thomson, Graeme James
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1994
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Abstract:
The reaction of the E.coli chaperonin GroEL (cpn 60) with the ATP analogue 2', 3' oxidised ATP (oATP) was studied with a view to identify the important amino acid(s) present at the ATP binding site of GroEL. Treatment with the reagent leads to loss of the ATPase activity of GroEL in a pseudo-first order fashion; this can be prevented by inclusion of ATP in the reaction mixture. Measurements of the stoichiometry of the reaction indicate that the loss of activity corresponds to the incorporation of about one oATP per subunit of GroEL. From analysis of the sequences of modified peptides it is proposed that the reaction probably occurs with one or both of the two cysteines Cys 457 and Cys 518, although the instability of the adduct(s) makes a definite identification of the site(s) of reaction difficult. The involvement of Cys side chains in the reaction with oATP was confirmed by using DTNB (5,5'-dithiobis(2-nitrobenzoate)) to estimate thiol groups in both modified and unmodified GroEL. The unfolding of GroEL in solutions in guanidinium chloride (GdnHCl) was also studied. From the results of CD, fluorescence and light scattering it is clear that major structural transitions in the protein occur over the range 1.0 - 1.5 M-GdnHCl, whilst the ATPase activity is lost at lower concentrations (0.75 M). The unfolding of the protein appears to be a highly cooperative process. After denaturation in concentrations of GdnHCl above 1.0 M, removal of the denaturing agent by dialysis results in the very nearly complete regain of secondary structure (as judged by CD) but not the regain of correct tertiary structure or quaternary structure, nor ATPase activity. The product was shown to be very sensitive to proteolysis by thermolysin, unlike the native protein, but did not show enhanced binding of ANS, a characteristic property of the 'molten globule' state of proteins.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.796937  DOI: Not available
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