Partitioning the effects of changes in a protein to the folded or unfolded forms by using a thermodynamic cycle: a change in Escherichia coli thioredoxin does not affect the unfolded state.


Abstract

Previously, we have introduced a method whereby novel disulfide side chains can be produced in the interior of a protein by modifying a cysteine residue after denaturant-induced unfolding [Wynn, R., & Richards, F. M. (1993) Proteins: Struct., Funct., Genet. 2, 395-403]. Here the disulfide exchange equilibrium constant, Kred, between the protein C32S,C35S-L78C thioredoxin and 2-hydroxyethyl disulfide is studied as a function of urea concentration. Since the disulfide exchange reaction and the stability of the proteins are thermodynamically linked, independent knowledge of the stabilities of the modified and unmodified proteins along with measurements of Kred allows us to assign Kred values for the folded and urea-unfolded forms of the protein. We find that the disulfide exchange reaction is more favorable for the folded protein, in agreement with the increased stability of the modified protein, and that the Kred values for both states are independent of the urea concentration. Finally, Kred values for the unfolded protein are the same, within experimental error, as that for N-acetylcysteine methylamide, an analog of cysteine in a peptide chain without the possibility of intramolecular interactions. Thus, we conclude that modification of position 78 of thioredoxin does not affect the unfolded state. The relevance of these results toward protein stability studies is discussed. Study holds ProTherm entries: 4630, 4631 Extra Details: additive : EDTA(1 mM),reduced protein thermodynamic; disulfide side chains; disulfide exchange reaction,intramolecular interactions

Submission Details

ID: UPLjjgV43

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:26 p.m.

Version: 1

Publication Details
Wynn R;Richards FM,Biochemistry (1993) Partitioning the effects of changes in a protein to the folded or unfolded forms by using a thermodynamic cycle: a change in Escherichia coli thioredoxin does not affect the unfolded state. PMID:8251515
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
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
5IKN 2016-03-03T00:00:00+0000 4.8 Crystal Structure of the T7 Replisome in the Absence of DNA
1F6M 2000-06-22T00:00:00+0000 2.95 CRYSTAL STRUCTURE OF A COMPLEX BETWEEN THIOREDOXIN REDUCTASE, THIOREDOXIN, AND THE NADP+ ANALOG, AADP+
1KEB 2001-11-15T00:00:00+0000 1.8 Crystal Structure of Double Mutant M37L,P40S E.coli Thioredoxin
1M7T 2002-07-22T00:00:00+0000 0 Solution Structure and Dynamics of the Human-Escherichia coli Thioredoxin Chimera: Insights into Thermodynamic Stability
1OAZ 2003-01-21T00:00:00+0000 2.78 IgE Fv SPE7 complexed with a recombinant thioredoxin
1SKR 2004-03-05T00:00:00+0000 2.4 T7 DNA Polymerase Complexed To DNA Primer/Template and ddATP
1SKS 2004-03-05T00:00:00+0000 2.3 Binary 3' complex of T7 DNA polymerase with a DNA primer/template containing a cis-syn thymine dimer on the template
1SKW 2004-03-05T00:00:00+0000 2.3 Binary 3' complex of T7 DNA polymerase with a DNA primer/template containing a disordered cis-syn thymine dimer on the template

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