ETD Collection

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Author: search.filters.author.Parbhoo, NishalSubject: search.filters.subject.Thioredoxin.

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    Topologically conserved hydrophobic residues of the thioredoxin C-subdomain stabilise GSTs
    (2014-06-12) Parbhoo, Nishal
    The thioredoxin-like fold is a well conserved fold that is present in many families of proteins. One such superfamily of proteins include the GSTs which are involved in phase II detoxification. GSTs primarily catalyse the metabolism of xenobiotics but are also involved in transporting non–substrate ligands and reactive compounds. The GST fold comprises an N-terminal thioredoxin domain and an all alpha helical C-terminal domain and is present in at least 18 classes of proteins. The N-terminal thioredoxin domain is characterised by the βαβαββα topology and can be further divided into two structural motifs, an N-terminal (βαβ) and a C-terminal (αββα) motif. A well conserved hydrophobic network exists between these two motifs and the role of the C-terminal motif is elucidated in this study using class Alpha GST as a model protein. A topologically conserved valine (Val58) and an isoleucine (Ile75) located on β3 and α3, respectively, were mutated to alanine. Secondary and tertiary structural characterisation as well as ligandin function of the mutant enzymes displayed no major structural alteration with respect to the wild-type enzyme. This was confirmed with high resolution crystal structures obtained. Enzymatic activity was maintained indicating that no structural alterations have occured that affects the active site dynamics and the domain interface as a result of the induced mutations. Thermal denaturation studies, however, indicated a slight destabilisation in the enzyme in the case of the valine mutation, but a large destabilisation was witnessed as a result of the isoleucine mutation. This is further observed in denaturant-induced equilibrium studies where the thermodynamic stability of proteins can be determined. Furthermore, as a result of the isoleucine mutation, the enzyme unfolds via a populated intermediate in contrast to the wild-type which globally unfolds via a two-state mechanism with no stable intermediates being populated. Pulse-proteolysis was employed as an additional probe for thermodynamic stability where the enzyme was digested by thermolysin at varying denaturant concentrations. Pulse-proteolysis results were in agreement with the thermal and denaturant-induced stability studies further confirming that the isoleucince substitution causes a large destabilisation. Thus these conserved hydrophobic residues of the thioredoxin C-subdomain play a crucial stabilising role in the GST fold.