Abstract
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 DeltatigDeltadnaK cells and prevented global protein misfolding at temperatures between 20 and 34 degrees C in these cells. We conclude that the PPIase activity is not required for the function of TF in folding of newly synthesized proteins.
Highlights
Trigger Factor (TF)1 was first discovered in Escherichia coli as a protein that triggered the translocation of the precursor of pro-OmpA into membrane vesicles [1]
The TF mutant proteins were analyzed for their structural integrity by circular dichroism measurements, proteinase K digestion, and determination of their ability to associate with E. coli ribosomes
TF F198A Is Deficient in peptidyl-prolyl cis/trans isomerase (PPIase) Activity—We tested the TF mutants for their catalytic PPIase activity toward two substrates, a chromogenic tetrapeptide and the model protein substrate RNase T1
Summary
Growth Conditions—Strains were grown in LB (Luria Broth) medium containing IPTG as indicated and supplemented with ampicillin (100 g/ml), tetracycline (5 g/ml), or kanamycin (40 g/ml) when appropriate. The dnaK gene of MC4100 was replaced with a kanamycin resistance cassette using plasmid pKD4 as PCR-template and the primers dnaK-5Ј (5Ј-CAGACTCACAACCACATGATGACCGAATATATAGTGGAGACGTTTAGGTGTAGGCTGGAGCTGCTTCG-3Ј) and dnaK-3Ј (5Ј-TTCCGCTGTTTTGGAAACGCCTAAAATCTCGTAATAATCTTGCTTAGCCATATGAATATCCTCCTTAG-3Ј) as described [20], resulting in strain GK1. The deletion of tig, encoding TF, was introduced by P1 transduction using a P1 lysate derived from a strain carrying ⌬tig::kan genetically linked to zba-3054::Tn10, conferring tetracycline resistance as described [7]. Upon 50-fold dilution of the denatured enzyme into GAPDH buffer (0.1 M potassium phosphate, pH 7.5, 1 mM EDTA, 5 mM dithiothreitol), aggregation was monitored by 90° light scattering at 620 nm in a spectrofluorometer (PerkinElmer Life Sciences; LS 55) in the presence or absence of 1 or 2 M TF or TF variants. Isolation of aggregates was performed as described previously [28]
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