Trigger Factor Peptidyl-prolyl cis/trans Isomerase Activity Is Not Essential for the Folding of Cytosolic Proteins in Escherichia coli

The ribosome-associated Trigger Factor (TF) cooperates with the DnaK system to assist the folding of newly synthesized polypeptides in Escherichia coli . TF unifies two functions in one to promote proper protein folding in vitro . First, as a chaperone it binds to unfolded protein substrates, thereby preventing aggregation and supporting productive folding. Second, TF catalyzes the cis/trans isomerization of peptidyl-prolyl bonds, which can be a rate-limiting step in protein folding. Here, we investigated whether the peptidyl-prolyl cis/trans isomerase (PPIase) function is essential for the folding activity of TF in vitro and in vivo by separating these two TF activities through site-directed mutagenesis of the PPIase catalytic center. Of the four different TF variants carrying point mutations in the PPIase domain, only the exchange of the conserved residue Phe-198 to Ala (TF F198A) abolished the PPIase activity of TF toward both a tetrapeptide and the model protein substrate RNase T1 in vitro . In contrast, all other activities of TF F198A tested were comparable with wild type TF. TF F198A retained a similar binding specificity toward membrane-bound peptides, assisted the refolding of denatured d -glyceraldehyde-3-phosphate dehydrogenase in vitro , and associated with nascent polypeptides in an in vitro transcription/translation system. Importantly, expression of the TF F198A encoding gene complemented the synthetic lethality of Δ tig Δ dnaK cells and prevented global protein misfolding at temperatures between 20 and 34 °C in these cells. We conclude that the PPIase activity is not required for the function of TF in folding of newly synthesized proteins.