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

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 (Cys247) 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.