Equilibrium and kinetic measurements of the conformational transition of reduced thioredoxin.


The single disulfide bond in Escherichia coli thioredoxin was reduced by reaction with a 20-fold excess of reduced dithiothreitol at neutral pH and 25 degrees C. For some measurements, reduced thioredoxin was further reacted with iodoacetamide to alkylate the cysteinyl residues. The denaturation transitions of oxidized, reduced, and reduced alkylated thioredoxin were observed by using far-ultraviolet circular dichroic and exclusion chromatographic measurements. Cleavage of the disulfide bond lowers the stability of the native thioredoxin to denaturation by about 2.4 kcal/mol, and subsequent alkylation lowers the stability by a further 1.6 kcal/mol. The kinetics of the conformational change of reduced thioredoxin in guanidine hydrochloride were observed by using exclusion chromatography at moderate pressure and 2 degrees C. Analyses of single and multimixing protocols are consistent with a predominant nonnative configuration in the denatured state and the transient accumulation of a compact nativelike intermediate during refolding. The intermediate can incorporate the nonnative configuration and can accommodate its isomerization. No compelling chromatographic evidence was found for a conformation having an elution time different from that characteristic for either the native or the denatured protein. Study holds ProTherm entries: 3871, 3872, 3873, 3874 Extra Details: conformational transition; alkylation; nonnative configuration;,isomerization

Submission Details

ID: hcGV3yvw

Submitter: Connie Wang

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

Version: 1

Publication Details
Kelley RF;Shalongo W;Jagannadham MV;Stellwagen E,Biochemistry (1987) Equilibrium and kinetic measurements of the conformational transition of reduced thioredoxin. PMID:3552046
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 Thioredoxin 1 P0AA27 THIO_ECO57
100.0 Thioredoxin 1 P0AA26 THIO_ECOL6
100.0 Thioredoxin 1 P0AA25 THIO_ECOLI
100.0 Thioredoxin 1 P0AA29 THIO_SALTI
100.0 Thioredoxin 1 P0AA28 THIO_SALTY
100.0 Thioredoxin 1 P0AA30 THIO_SHIFL