Recognition of protein substrates by the prolyl isomerase trigger factor is independent of proline residues 1 1Edited by P. E. Wright

Schölz, Christian; Mücke, Matthias; Rapé, Michael; Pecht, Michael; Pahl, Andreas; Bang, Holger; Schmid, Franz X.

doi:10.1006/jmbi.1997.1604

Cited by 46 publications

(36 citation statements)

References 52 publications

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“…TF exhibits PPIase activity and is known to catalyze the PPIase-dependent refolding of RCM-T1 (a reduced and carboxymethylated variant of RNase T 1 ) in vitro (12,14). On the other hand, the binding of TF to some protein substrates has been reported to be independent of proline residues (13). Whether the PPIase activity is needed for the chaperonelike activity of TF found in this study has not been determined.…”

Section: Discussionmentioning

confidence: 73%

Overexpression of Trigger Factor Prevents Aggregation of Recombinant Proteins in Escherichia coli

Nishihara

Kanemori

Yanagi

et al. 2000

Appl Environ Microbiol

272

186

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To examine the effects of overexpression of trigger factor (TF) on recombinant proteins produced in Escherichia coli, we constructed plasmids that permitted controlled expression of TF alone or together with the GroEL-GroES chaperones. The following three proteins that are prone to aggregation were tested as targets: mouse endostatin, human oxygen-regulated protein ORP150, and human lysozyme. The results revealed that TF overexpression had marked effects on the production of these proteins in soluble forms, presumably through facilitating correct folding. Whereas overexpression of TF alone was sufficient to prevent aggregation of endostatin, overexpression of TF together with GroEL-GroES was more effective for ORP150 and lysozyme, suggesting that TF and GroEL-GroES play synergistic roles in vivo. Although coexpression of the DnaK-DnaJGrpE chaperones was also effective for endostatin and ORP150, coexpression of TF and GroEL-GroES was more effective for lysozyme. These results attest to the usefulness of the present expression plasmids for improving protein production in E. coli.Recombinant proteins produced in Escherichia coli often aggregate or degrade rapidly because of their inability to form correct tertiary structures due to anomalies in protein folding. In some cases, overexpression of molecular chaperones, such as GroEL-GroES and DnaK-DnaJ-GrpE, facilitate protein folding and enhance production of active proteins (16,18). We previously constructed a series of versatile plasmids for controlled expression of these chaperones and described their utility for stabilizing or preventing aggregation of certain recombinant proteins (9). However, these plasmids are not always effective; there are many instances in which production of active proteins is not improved by the chaperones and seems to require additional factors or conditions. Thus, we examined new chaperonelike factors, including E. coli trigger factor (TF), whose role in protein folding was implicated by the results of several in vitro experiments. This 50-kDa protein exhibits peptidyl-prolyl cis/trans isomerase (PPIase) activity and has a domain structure that is conserved in the FK506-binding protein family (14,17). TF was originally identified as a protein that can bind to certain precursor proteins and facilitate their transport into membrane vesicles (1). The results of subsequent studies, however, failed to support hypothesis that TF plays a role in protein transport (2) and instead suggested that it may play a role in protein folding because of its association with nascent polypeptides, as well as the 50S ribosome (3, 17). Moreover, TF was found to be associated with GroEL and can strengthen GroEL-substrate binding to facilitate protein folding (5) or degradation (6).In this study, we constructed plasmids with which we could easily manipulate the production of TF, alone or together with GroEL-GroES chaperones, and examined the effects of coexpression of these molecules on the production of several recombinant proteins. The target proteins employed...

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Section: Discussionmentioning

confidence: 73%

Overexpression of Trigger Factor Prevents Aggregation of Recombinant Proteins in Escherichia coli

Nishihara

Kanemori

Yanagi

et al. 2000

Appl Environ Microbiol

272

186

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“…It consists of a ribosome-binding domain, a prolyl isomerase domain of the FKBP type, and a chaperone domain (Fig. 1B), and it catalyzes proline-limited protein folding reactions in vitro very well (23,49,50). Like the other FKBPs, trigger factor shows a high substrate specificity toward Xaa-Pro bonds in tetrapeptides (Table 1) and, in its activity profile (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The dissociation constants of the complexes formed between trigger factor or SlyD and non-native proteins, the K M values for catalyzed folding, and the K I values for their inhibition by permanently unfolded proteins are all in the range of 1 M (49,50). This suggests that a well balanced affinity is important for the proper function of the chaperone domains of folding enzymes.…”

Section: Discussionmentioning

confidence: 98%

Chaperone domains convert prolyl isomerases into generic catalysts of protein folding

Jakob

Žoldák

Aumüller

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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The cis/trans isomerization of peptide bonds before proline (prolyl bonds) is a rate-limiting step in many protein folding reactions, and it is used to switch between alternate functional states of folded proteins. Several prolyl isomerases of the FK506-binding protein family, such as trigger factor, SlyD, and FkpA, contain chaperone domains and are assumed to assist protein folding in vivo. The prolyl isomerase activity of FK506-binding proteins strongly depends on the nature of residue Xaa of the Xaa-Pro bond. We confirmed this in assays with a library of tetrapeptides in which position Xaa was occupied by all 20 aa. A high sequence specificity seems inconsistent with a generic function of prolyl isomerases in protein folding. Accordingly, we constructed a library of protein variants with all 20 aa at position Xaa before a rate-limiting cis proline and used it to investigate the performance of trigger factor and SlyD as catalysts of proline-limited folding. The efficiencies of both prolyl isomerases were higher than in the tetrapeptide assays, and, intriguingly, this high activity was almost independent of the nature of the residue before the proline. Apparently, the almost indiscriminate binding of the chaperone domain to the refolding protein chain overrides the inherently high sequence specificity of the prolyl isomerase site. The catalytic performance of these folding enzymes is thus determined by generic substrate recognition at the chaperone domain and efficient transfer to the active site in the prolyl isomerase domain.folding catalysis ͉ folding helpers ͉ folding mechanism ͉ SlyD ͉ trigger factor T he cis/trans isomerization of peptide bonds before proline (Xaa-Pro or prolyl bonds; Fig. 1A) is an intrinsically slow reaction that depends on the nature of the amino acid Xaa (1-3). It determines the rates of many protein folding reactions (4-6), is used as a molecular switch (7-16), and is catalyzed by prolyl isomerases (17-21). Most of the prolyl isomerases that assist in cellular protein folding contain catalytic domains that are homologous to human FKBP12 (FK-506 binding protein of 12 kDa; Fig. 1B) and chaperone domains, which interact transiently with non-native proteins (Fig. 1B). The trigger factor (22-25) and SlyD [product of the slyD (sensitive-to lysis) gene, a cytosolic Escherichia coli chaperone] (26-29) belong to this family of folding enzymes. The chaperone domain of SlyD (the ''insertin-flap'' or IF domain) shows a unique fold (30) and is structurally not related to other chaperones. The chaperone domain of trigger factor shows similarities with prefoldin (31)and SurA (32). FkpA (periplasmic FKBP of E. coli) (33-35) of prokaryotes and FKBP52 of eukaryotes (36) display similar combinations of prolyl isomerase and chaperone domains.FKBP-type prolyl isomerases are highly specific with regard to residue Xaa before the proline (37-39). An initial characterization of human FKBP12 with various proline-containing tetrapeptides and a protease-coupled assay (17) indicated that it catalyzes isomerizati...

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“…The k cat /K m of Mip corresponds to the value found for the hFKBP12 (k cat /K m ϭ 1.2 ϫ 10 Ϫ4 M Ϫ1 s Ϫ1 ) (52), and it is likely that the reduced folding efficiency of Mip originates in the loss of the dimeric state as well as of residues involved in substrate binding. In analogy, it has been shown that the high affinity of the E. coli trigger factor for unfolded protein substrates can be attributed to the N-and C-terminal domains which encompass the FKBP-like domain (45,46,52). Another example is the chaperone-like activity of E. coli FkpA, which requires the PPIase active site and the full-length structure of the protein during the refolding of the maltose-binding protein MalE31 (3,37).…”

Section: Discussionmentioning

confidence: 99%

Biochemical and Functional Analyses of the Mip Protein: Influence of the N-Terminal Half and of Peptidylprolyl Isomerase Activity on the Virulence of Legionella pneumophila

et al. 2003

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Recognition of protein substrates by the prolyl isomerase trigger factor is independent of proline residues 1 1Edited by P. E. Wright

Cited by 46 publications

References 52 publications

Overexpression of Trigger Factor Prevents Aggregation of Recombinant Proteins in Escherichia coli

Overexpression of Trigger Factor Prevents Aggregation of Recombinant Proteins in Escherichia coli

Chaperone domains convert prolyl isomerases into generic catalysts of protein folding

Biochemical and Functional Analyses of the Mip Protein: Influence of the N-Terminal Half and of Peptidylprolyl Isomerase Activity on the Virulence of Legionella pneumophila

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