Mutagenic analysis in a pure molecular system shows that thioredoxin-interacting protein residue Cys247 is necessary and sufficient for a mixed disulfide formation with thioredoxin

Protein Sci. 2012 Sep;21(9):1323-33. doi: 10.1002/pro.2119. Epub 2012 Aug 9.


The human thioredoxin (TRX)-interacting protein is found in multiple subcellular compartments and plays a major role in redox homeostasis, particularly in the context of metabolism (e.g., lipidemia and glycemia) and apoptosis. A molecular approach to the protein's modus operandi is still needed because some aspects of the TRX-interacting protein-mediated regulation of TRX are not clearly understood. To this end, His-tagged TRX-interacting proteins were over-expressed in Escherichia coli. Because the protein is expressed mainly in inclusion bodies, it was denatured in high concentrations of guanidium hydrochloride, centrifuged, and purified by Ni-NTA affinity chromatography. His-TRX-interacting protein was then refolded by dialysis and its restructuring monitored by circular dichroism spectrometry. This preparation resulted in the formation of a covalent complex with recombinant human TRX, demonstrating that association occurs without the intervention of other partner proteins. Multiple cysteine-to-serine mutants of TRX-interacting protein were produced and purified. These mutations were efficient in limiting the formation of disulfide-linked homo-oligomers in an oxidizing environment. The mutants were also used to gain functional insight into the formation of the TRX-interacting protein-TRX complexes. These complexes were able to form in the absence of internal disulfide bridges. A mutant with all but one cysteine changed to serine (Cys ²⁴⁷) also showed an enhanced capacity to form complexes with TRX demonstrating, in a pure molecular system, that this particular cysteine is likely responsible for the disulfide bridge between TRX-interacting protein and TRX.

MeSH terms

  • Amino Acid Substitution
  • Animals
  • Carrier Proteins / chemistry
  • Carrier Proteins / genetics*
  • Carrier Proteins / isolation & purification
  • Carrier Proteins / metabolism*
  • Cell Line
  • Cloning, Molecular
  • Cysteine / chemistry
  • Cysteine / genetics
  • Cysteine / metabolism
  • Disulfides / metabolism*
  • Escherichia coli / genetics
  • Humans
  • Oxidation-Reduction
  • Protein Refolding
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Thioredoxins / metabolism*


  • Carrier Proteins
  • Disulfides
  • Recombinant Proteins
  • TXN protein, human
  • TXNIP protein, human
  • Thioredoxins
  • Cysteine