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

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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.

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