The conformational stability of a detoxification enzyme widely used as a fusion-protein affinity tag.
Date
1997
Authors
Kaplan, Warren H
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
A glutathione S-transferase (Sj26GST) from Schistosoma japonicum, which functions in
the parasite's Phase II detoxification pathway, is expressed by the Pharmacia pGEX-2T
plasmid and is widely used as a fusion-protein affinity tag. It contains all 217 residues
of Sj26GST and an ad titional 9-residue peptide linker with a thrombin cleavage site at
its C-terminus. Size-exclusion HPLC (SEC-HPLC) and SDS-PAGE studies indicate that
purification of the homodimeric protein under nonreducing conditions results in the
reversible for-ration of significant amounts of 160 -kDa and larger aggregates without
a loss in catalytic activity. The basis for oxidative aggregation can be ascribed to the high
degree of exposure of the four cysteine residues per subunit. The conformational stability
of the dimeric protein was studied by urea- and temperature-induced unfolding
techniques. Fluorescence-spectroscopy, SEC-HPLC, urea- and temperature-gradient gel
electrophoresis, ultraviolet melting, differential scanning micro calorimetry , and enzyme
activity were employed to monitor structural and functional changes. The unfolding data
indicate the absence of thermodynamically stable intermediates and that the
umolding/refolding transition is a two-state process involving folded native dimer and
unfolded monomer. The stability of the protein was found to be dependent on its
concentration with a ~GO(H20) = 26 ±1.7 kcal/mol. The conformational stability was
unchanged in the presence of the leading antischistosomal drug Praziquantel, which
bound the protein with a Kd = 9 ±1.8 p,M. The strong relationship observed between
the m-v,llue and the size of the protein indicates that the amount of protem. surface
exposed to solvent upon unfolding is the major structural de.erminant for the dependence
of the protein's free energy of unfolding on urea concentration. 'Ihermograms obtained
by differential scanning calorimetry also fitted to a two-state irreversible unfolding
transition, both in the presence and absence of Praziquantel, with values of ~Cp = 1779
cal mol-IK-I
, ~HcaI = 227 kcal/mol, AHVH ::::::233 kcal/mol (r :::::~:HVHIAlIcal = 1.02)
and AS = 354 cal mol''K". The low ~Cp and ~S, when compared with the theoretically
determined values, implied that the thermal denaturation of Sj26GST did not result in
complete unfolding of the protein,
Description
A thesis submitted to the Faculty of Science, University of the Witwatersrand, in
fulfilment of the requirements for the degree of Doctor of Philosophy.
Keywords
Glutathione transferase., Enzymatic analysis., Proteins., Biochemistry.