Abstract
The conserved fold of thioredoxin (Trx)-like thiol/disulfide oxidoreductases contains an invariant cis-proline residue (P76 in Escherichia coli Trx) that is essential for Trx function and that is responsible for the folding rate-limiting step. E. coli Trx contains four additional prolines, which are all in the trans conformation in the native state. Notably, a recent study revealed that replacement of all four trans prolines in Trx by alanines (Trx variant Trx1P) further slowed the rate-limiting step 25-fold, indicating that one or several of the four trans prolines accelerate the trans-to-cis transition of P76 in Trx wild-type (wt). Here, we characterized the folding kinetics of Trx variants containing cisP76 and one or several of the natural trans prolines of Trx wt with NMR spectroscopy. First, we demonstrate that the isomerization reaction in Trx1P is a pure two-state transition between two distinct tertiary structures, in which all observed NMR resonances changes follow the same first-order kinetics. Moreover, we show that trans-P68 is the critical residue responsible for the faster folding of wt Trx relative to the single-proline (P76) variant Trx1P, as the two-proline variant Trx2P(P76P68) already folds seven times faster than Trx1P. trans-P34 also accelerates trans-to-cis isomerization of P76, albeit to a smaller extent. Overall, the results demonstrate that trans prolines can significantly modulate the kinetics of rate-limiting trans-to-cis proline isomerization in protein folding. Finally, we discuss possible mechanisms of acceleration and the potential significance of a protein-internal folding acceleration mechanism for Trx in a living cell.