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 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 PDB Entries

Structure ID Release Date Resolution Structure Title
2LEG 2011-10-26 Membrane protein complex DsbB-DsbA structure by joint calculations with solid-state NMR and X-ray experimental data
1A23 1998-09-16 SOLUTION NMR STRUCTURE OF REDUCED DSBA FROM ESCHERICHIA COLI, MINIMIZED AVERAGE STRUCTURE
1A24 1998-09-16 SOLUTION NMR STRUCTURE OF REDUCED DSBA FROM ESCHERICHIA COLI, FAMILY OF 20 STRUCTURES
2NDO 2017-02-08 Structure of EcDsbA-sulfonamide1 complex
1FVK 1997-08-20 1.7 THE 1.7 ANGSTROM STRUCTURE OF WILD TYPE DISULFIDE BOND FORMATION PROTEIN (DSBA)
4ZIJ 2016-05-11 1.78 Crystal structure of E.Coli DsbA in complex with 2-(4-iodophenylsulfonamido) benzoic acid
3DKS 2009-05-12 1.9 DsbA substrate complex
1ACV 1997-10-15 1.9 DSBA MUTANT H32S
2B3S 2006-09-05 1.96 structure of the DSBA mutant (P31G-C33A)
6BQX 2017-12-27 1.99 Crystal structure of Escherichia coli DsbA in complex with N-methyl-1-(4-phenoxyphenyl)methanamine
6BR4 2017-12-27 1.99 Crystal structure of Escherichia coli DsbA in complex with {N}-methyl-1-(3-thiophen-2-ylphenyl)methanamine
1AC1 1997-10-15 2.0 DSBA MUTANT H32L
1U3A 2005-05-03 2.0 mutant DsbA
1A2J 1998-09-16 2.0 OXIDIZED DSBA CRYSTAL FORM II
1DSB 1994-01-31 2.0 CRYSTAL STRUCTURE OF THE DSBA PROTEIN REQUIRED FOR DISULPHIDE BOND FORMATION IN VIVO
1FVJ 1997-05-15 2.06 THE 2.06 ANGSTROM STRUCTURE OF THE H32Y MUTANT OF THE DISULFIDE BOND FORMATION PROTEIN (DSBA)
1UN2 2003-09-26 2.4 Crystal structure of circularly permuted CPDSBA_Q100T99: Preserved Global Fold and Local Structural Adjustments
4TKY 2015-01-14 2.5 The complex structure of E. coli DsbA bound to a peptide at the DsbA/DsbB interface
1TI1 2005-05-03 2.6 crystal structure of a mutant DsbA
2B6M 2006-10-17 2.65 Structure of the DsbA mutant (P31A-C33A)
1A2L 1998-07-08 2.7 REDUCED DSBA AT 2.7 ANGSTROMS RESOLUTION
1A2M 1998-07-08 2.7 OXIDIZED DSBA AT 2.7 ANGSTROMS RESOLUTION, CRYSTAL FORM III
1BQ7 1999-08-20 2.8 DSBA MUTANT P151A, ROLE OF THE CIS-PROLINE IN THE ACTIVE SITE OF DSBA
2HI7 2006-12-05 3.7 Crystal structure of DsbA-DsbB-ubiquinone complex
2ZUP 2009-04-14 3.7 Updated crystal structure of DsbB-DsbA complex from E. coli
3E9J 2008-11-25 3.7 Structure of the charge-transfer intermediate of the transmembrane redox catalyst DsbB

Relevant UniProtKB Entries

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