Urea dependence of thiol-disulfide equilibria in thioredoxin: confirmation of the linkage relationship and a sensitive assay for structure.


Abstract

Thioredoxin contains a single disulfide bond that can be reduced without perturbing significantly the structure of the enzyme. Upon reduction of the disulfide, protein stability decreases. We have experimentally tested the expected linkage relationship between disulfide bond formation and protein stability for thioredoxin. In order to do this, it is necessary to measure the equilibrium constant for disulfide bond formation in both the folded and unfolded states of the protein. Using glutathione as a reference species, we have measured the equilibrium constant for forming the disulfide bond (effective concentration) in thioredoxin as a function of urea concentration. As a control, we show that urea per se does not interfere with our measurements of thiol-disulfide equilibrium constants. Comparison of the values obtained for disulfide bond formation in the folded and unfolded states with the free energies for unfolding oxidized and reduced thioredoxin using circular dichroism confirms the expected linkage relationship. The urea dependence of thiol-disulfide equilibria provides a sensitive assay for folded structure in peptides or proteins. The method should also be useful to evaluate the stabilizing or destabilizing effect of natural or genetically engineered disulfides in proteins. In future work, the effects of amino acid substitutions on disulfide bond formation could be evaluated individually in the native and unfolded states of a protein. Study holds ProTherm entries: 3842, 3843 Extra Details: additive : EDTA(1 mM), disulfide bond formation; equilibrium constant;,sensitive assay; folded structure

Submission Details

ID: o4tFC3tQ

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:23 p.m.

Version: 1

Publication Details
Lin TY;Kim PS,Biochemistry (1989) Urea dependence of thiol-disulfide equilibria in thioredoxin: confirmation of the linkage relationship and a sensitive assay for structure. PMID:2669972
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
5IKN 2016-03-03T00:00:00+0000 4.8 Crystal Structure of the T7 Replisome in the Absence of DNA
3DXB 2008-07-24T00:00:00+0000 2.2 Structure of the UHM domain of Puf60 fused to thioredoxin
5E4W 2015-10-07T00:00:00+0000 2.8 Crystal structure of cpSRP43 chromodomains 2 and 3 in complex with the Alb3 tail
1T8E 2004-05-12T00:00:00+0000 2.54 T7 DNA Polymerase Ternary Complex with dCTP at the Insertion Site.
1TKD 2004-06-08T00:00:00+0000 2.49 T7 DNA polymerase ternary complex with 8 oxo guanosine and dCMP at the elongation site
1TK0 2004-06-07T00:00:00+0000 2.3 T7 DNA polymerase ternary complex with 8 oxo guanosine and ddCTP at the insertion site
1X9M 2004-08-23T00:00:00+0000 2.1 T7 DNA polymerase in complex with an N-2-acetylaminofluorene-adducted DNA
2TRX 1990-03-19T00:00:00+0000 1.68 CRYSTAL STRUCTURE OF THIOREDOXIN FROM ESCHERICHIA COLI AT 1.68 ANGSTROMS RESOLUTION
1X9S 2004-08-24T00:00:00+0000 2.7 T7 DNA polymerase in complex with a primer/template DNA containing a disordered N-2 aminofluorene on the template, crystallized with dideoxy-CTP as the incoming nucleotide.
1ZCP 2005-04-12T00:00:00+0000 2.3 Crystal Structure of a catalytic site mutant E. coli TrxA (CACA)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Thioredoxin 1 P0AA30 THIO_SHIFL
100.0 Thioredoxin 1 P0AA28 THIO_SALTY
100.0 Thioredoxin 1 P0AA29 THIO_SALTI
100.0 Thioredoxin 1 P0AA25 THIO_ECOLI
100.0 Thioredoxin 1 P0AA26 THIO_ECOL6
100.0 Thioredoxin 1 P0AA27 THIO_ECO57