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


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

Submission Details

ID: U7bKHhs83

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:32 p.m.

Version: 1

Publication Details
Moutiez M;Burova TV;Haertlé T;Quéméneur E,Protein Sci. (1999) On the non-respect of the thermodynamic cycle by DsbA variants. PMID:10210189
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Thiol:disulfide interchange protein DsbA P0AEG5 DSBA_ECO57
100.0 Thiol:disulfide interchange protein DsbA P0AEG4 DSBA_ECOLI
99.5 Thiol:disulfide interchange protein DsbA P0A4L6 DSBA_ECO27
99.5 Thiol:disulfide interchange protein DsbA P0A4L5 DSBA_ECOL6
99.5 Thiol:disulfide interchange protein DsbA P52235 DSBA_SHIFL