Acceleration of the Rate-Limiting Step of Thioredoxin Folding by Replacement of its Conserved cis-Proline with (4 S)-Fluoroproline.

The incorporation of the non-natural amino acids (4R)- and (4S)-fluoroproline (Flp) has been successfully used to improve protein stability, but little is known about their effect on protein folding kinetics. Here we analyzed the influence of (4R)- and (4S)-Flp on the rate-limiting trans-to-cis isomerization of the Ile75-Pro76 peptide bond in the folding of Escherichia coli thioredoxin (Trx). While (4R)-Flp at position 76 had essentially no effect on the isomerization rate in the context of the intact tertiary structure, (4S)-Flp accelerated the folding reaction ninefold. Similarly, tenfold faster trans-to-cis isomerization of Ile75-(4S)-Flp76 relative to Ile75-Pro76 was observed in the unfolded state of Trx. Our results show that the replacement of cis prolines by non-natural proline analogues can be used for modulating the folding rates of proteins with cis prolyl-peptide bonds in the native state.