The mechanism of the disulfide-bond forming enzyme DsbA depends on the very low pKa of a cysteine residue in its active-site and on the relative instability of the oxidized enzyme compared to the reduced one. A thermodynamic cycle has been used to correlate its redox properties to the difference in the free energies of folding (deltadeltaGred/ox) of the oxidized and reduced forms. However, the relation was proved unsatisfied for a number of DsbA variants. In this study, we investigate the thermodynamic and redox properties of a highly destabilized variant DsbA(P151A) (substitution of cis-Pro151 by an alanine) by the means of intrinsic tryptophan fluorescence and by high-sensitivity differential scanning calorimetry (HS-DSC). When the value of deltadeltaGred/ox obtained fluorimetrically for DsbA(P151A) does not correlate with the value expected from its redox potential, the value of deltadeltaGred/ox provided by HS-DSC are in perfect agreement with the predicted thermodynamic cycle for both wild-type and variant. HS-DSC data indicate that oxidized wild-type enzyme and the reduced forms of both wild-type and variant unfold according to a two-state mechanism. Oxidized DsbA(P151A) shows a deviation from two-state behavior that implies the loss of interdomain cooperativity in DsbA caused by Pro151 substitution. The presence of chaotrope in fluorimetric measurements could facilitate domain uncoupling so that the fluorescence probe (Trp76) does not reflect the whole unfolding process of DsbA(P151A) anymore. Thus, theoretical thermodynamic cycle is respected when an appropriate method is applied to DsbA unfolding under conditions in which protein domains still conserve their cooperativity. Study holds ProTherm entries: 6712, 6713, 6714, 6715, 6716, 6717, 6718, 6719, 6720, 6721, 6722, 6723 Extra Details: additive : EDTA(1 mM), calorimetry; DsbA; redox potential; stability;,themodynamic cycle
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:32 p.m.
|Number of data points||26|
|Proteins||Thiol:disulfide interchange protein DsbA ; Thiol:disulfide interchange protein DsbA|
|Assays/Quantities/Protocols||Experimental Assay: dG ; Experimental Assay: dG ; Experimental Assay: dCp ; Experimental Assay: dHcal ; Experimental Assay: Tm ; Experimental Assay: dHvH ; Derived Quantity: dTm|
|Libraries||Mutations for sequence AQYEDGKQYTTLEKPVAGAPQVLEFFSFFCPHCYQFEEVLHISDNVKKKLPEGVKMTKYHVNFMGGDLGKDLTQAWAVAMALGVEDKVTVPLFEGVQKTQTIRSASDIRDVFINAGIKGEEYDAAWNSFVVKSLVAQQEKAAADVQLRGVPAMFVNGKYQLNPQGMDTSNMDVFVQQYADTVKYLSEKK|