4-Thiaproline accelerates the slow folding phase of proteins containing cis prolines in the native state by two orders of magnitude

The cis/trans isomerization of peptidyl-prolyl peptide bonds is often the bottleneck of the refolding reaction for proteins containing cis proline residues in the native state. Proline (Pro) analogues, especially C4-substituted fluoroprolines, have been widely used in protein engineering to enhance the thermodynamic stability of peptides and proteins and to investigate folding kinetics. 4-thiaproline (Thp) has been shown to bias the ring pucker of Pro, to increase the cis population percentage of model peptides in comparison to Pro, and to diminish the activation energy barrier for the cis/trans isomerization reaction. Despite its intriguing properties, Thp has been seldom incorporated into proteins. Moreover, the impact of Thp on the folding kinetics of globular proteins has never been reported. In this study, we show that upon incorporation of Thp at cisPro76 into the thioredoxin variant Trx1P the half-life of the refolding reaction decreased from [~]2 hours to [~]35 seconds. A dramatic acceleration of the refolding rate could be observed also for the protein pseudo wild-type barstar upon replacement of cisPro48 with Thp. Quantum chemical calculations revealed that the replacement of the C{gamma}H2 group by a sulfur atom in the pyrrolidine ring, lowers the barrier for cis/trans rotation due to a weakened peptide bond. The protein variants retained their thermodynamic stability upon incorporation of Thp, while the catalytic and enzymatic activities of the modified Trx1P remained unchanged. Our results show that the Pro isostere Thp might eliminate the bottleneck of the refolding reaction of proteins containing cis proline residues in the native state, independent from the local structural environment.