Amino acid residues important for folding of thioredoxin are revealed only by study of the physiologically relevant reduced form of the protein.

Thioredoxin-1 (TrxA) of Escherichia coli is a cytoplasmic enzyme that maintains the cysteines of substrate proteins in the reduced form (3). It does this by utilizing one of its two redox-active cysteines (cysteine-32) to attack a substrate’s disulfide bonds, thus initiating the reductive process that leads to a reduced substrate and an oxidized thioredoxin (4). The enzyme thioredoxin reductase then transfers electrons from NADPH to thioredoxin, returning its cysteines to the reduced state. Because of its abundance and relative ease of purification, thioredoxin has been the subject of numerous in vitro protein folding studies (e.g. (5–10), etc). However, most studies of thioredoxin folding have been carried out on the oxidized form of thioredoxin because it is rapidly air oxidized in the absence of a reducing agent, because oxidized thioredoxin is more stable than the reduced form, and because oxidized thioredoxin folds with simplified kinetics. We previously described our use of a genetic screen to isolate new classes of mutants of thioredoxin that were defective for folding (11). When we examined their conformational stability and refolding kinetics in vitro, all but one of these folding mutants also showed significant defects in the refolding of the oxidized proteins. The exceptional mutant contained a relatively conservative substitution (aspartate to asparagine) at position 15, which is completely solvent exposed in structural models of thioredoxin and which had not been previously implicated in thioredoxin folding. Furthermore, the D15N mutant protein, which we presumed to cause a defect in thioredoxin folding in vivo, did not exhibit such a defect in preliminary in vitro studies on a reduced form of this mutant protein. Surprisingly, the D15N substitution had no effect on the rate of refolding of oxidized thioredoxin and even increased its conformational stability. In the present study, we revisited our genetic screen in order to isolate more thioredoxin folding mutants. We submitted several of the purified mutant proteins to a more rigorous analysis of their conformational dynamics and refolding kinetics. In addition, we carried out the first extensive folding analysis of the reduced form of thioredoxin and applied it to some of the mutant proteins. Among the mutations that we isolated were some that caused novel substitutions at D15 as well as substitutions at D13, which is also completely solvent exposed in the thioredoxin structure. These substitutions cause defects in the refolding kinetics of the reduced, but not the oxidized, forms of these proteins. We also find that a high proportion of the folding mutations affect either the penultimate amino acid, L107, or alter the chain-terminating codon causing extension of the carboxy-terminal amino acid sequence. These findings, along with a L107A mutant constructed here, strengthen previous evidence for a key role of L107 in folding and suggest that altering the carboxy-terminus of the protein in other ways may also interfere with folding (11). Our results demonstrate the importance of studying the physiologically relevant form of a protein in vitro and suggest that genetic studies of this sort may allow a significant narrowing down of candidates for key residues in folding.