A ribosome-associated peptidyl-prolyl cis/trans isomerase identified as the trigger factor.

Abstract

Peptidyl‐prolyl cis/trans isomerases (PPIases) are enzymes that catalyse protein folding both in vitro and in vivo. We isolated a peptidyl‐prolyl cis/trans isomerase (PPIase) which is specifically associated with the 50S subunit of the Escherichia coli ribosome. This association was abolished by adding at least 1.5 M LiCl. Sequencing the N‐terminal amino acids in addition to three proteolytic fragments totalling 62 amino acids revealed that this PPIase is identical to the E.coli trigger factor. A comparison of the amino acid sequence of trigger factor with those of other PPIase families shows little similarities, suggesting that trigger factor may represent an additional family of PPIases. Trigger factor was purified to homogeneity on a preparative scale from E.coli and its enzymatic properties were studied. In its activity towards oligopeptide substrates, the trigger factor resembles the FK506‐binding proteins (FKBPs). Additionally, the pattern of subsite specificities with respect to the amino acid preceding proline in Suc‐Ala‐Xaa‐Pro‐Phe‐4‐nitroanilides is reminiscent of FKBPs. However, the PPIase activity of the trigger factor was not inhibited by either FK506 or by cyclosporin A at concentrations up to 100 microM. In vitro, the trigger factor catalysed the proline‐limited refolding of a variant of RNase T1 much better than all other PPIases that have been examined so far.

An 11.8 kDa proteolytic fragment of the E. coli trigger factor represents the domain carrying the peptidyl‐prolyl cis/trans isomerase activity

“…Polyclonal antiserum against trigger factor was obtained from rabbit after immunisation with trigger factor fragments (Pab Productions, Herbertshausen, Germany). Trigger factor was isolated from E. coli cell extracts as described [6]. The 70S ribosomes were a kind gift from K. Nierhaus.…”

“…Further, TF was detected at the 70S ribosome dissociating in a saltdependent manner [5]. The binding was proved exclusively for the 50S subunit [5,6] known for covering the exit domain of the nascent polypeptide chain.…”

“…TF accelerates effectively the trans to cis isomerization of a model protein [6]. As was calculated via published methods about 5% of all peptidyl-prolyl bonds known in the spatial structure are in the cis conformation in native proteins [15,16].…”

“…The cyclophilins and FKBPs are characterised through their binding to cyclosporin A and FK506, respectively, thereby inhibiting their PPIase activity. The PPIase activity of TF was not affected at all by these effectors but its subsite specificity was found to be reminiscent of FKBPs [6]. A certain degree of sequence homology of an inner area of TF to FKBPs has also been reported [12].…”

“…Recently, it could be demonstrated that TF belongs to the peptidyl-prolyl cis/trans isomerases (PPIases) [6]. PPIases cat-*Corresponding author.…”