On the non-respect of the thermodynamic cycle by DsbA variants

Abstract

The mechanism of the disulfide-bond forming enzyme DsbA depends on the very low pK_a 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 (ΔΔG^red/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 ΔΔG^red/ox obtained fluorimetrically for DsbA_P151A does not correlate with the value expected from its redox potential, the value of ΔΔG^red/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.