Dynamic association of trigger factor with protein substrates

Maier, Raimund; Schölz, Christian; Schmid, Franz X.

doi:10.1006/jmbi.2000.5192

Cited by 69 publications

(59 citation statements)

References 36 publications

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“…In contrast, the presence of TF counteracted the proteolysis of all non-native arrested polypeptides tested. Consistent with the rapid mode of TF substrate binding and release, which is required for the efficient folding of proteins on the time scale of seconds up to minutes (29), the protection provided by TF was transient in all cases tested.…”

Section: Discussionsupporting

confidence: 62%

Trigger Factor Forms a Protective Shield for Nascent Polypeptides at the Ribosome

Hoffmann

Merz

Rutkowska

et al. 2006

Journal of Biological Chemistry

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In prokaryotes, the ribosome-associated Trigger Factor is the first chaperone newly synthesized polypeptides encounter when they emerge from the ribosomal exit tunnel. The effects that Trigger Factor exerts on nascent polypeptides, however, remain unclear. Here we analyzed the potential of the Trigger Factor to shield nascent polypeptides at the ribosome. A set of arrested nascent polypeptides differing in origin, size, and folding status were synthesized in an Escherichia coli-based in vitro transcription/translation system and tested for sensitivity to degradation by the unspecific protease proteinase K. In the absence of Trigger Factor, nascent polypeptides exposed outside the ribosomal exit tunnel were rapidly degraded unless they were folded into a compact domain. The presence of Trigger Factor, as well as a Trigger Factor fragment lacking its peptidyl-prolyl isomerase domain, counteracted degradation of all unfolded nascent polypeptides tested. This protective function was specific for ribosome-tethered Trigger Factor, since neither non-ribosomal Trigger Factor nor the DnaK system, which cooperates with Trigger Factor in the folding process in vivo, revealed a comparable efficiency in protection. Furthermore, shielding by Trigger Factor was not restricted to short stretches of nascent chains but was evident for large, non-native nascent polypeptides exposing up to 41 kDa outside the ribosome. We suggest that Trigger Factor supports productive de novo folding by shielding nascent polypeptides on the ribosome thereby preventing untimely degradation or aggregation processes. This protected environment provided by Trigger Factor might be particularly important for large multidomain proteins to fold productively into their native states.Cells employ a large arsenal of molecular chaperones to promote the folding of newly synthesized proteins in the cytosol (1-3). At the forefront are chaperones, which transiently bind to ribosomes and thereby are optimally positioned to associate with emerging nascent polypeptides. In eubacteria, Trigger Factor (TF) 3 binds to ribosomes near the exit of the peptide tunnel through major contacts to L23 (4 -7) and interacts co-translationally with nascent polypeptides (8, 9). Downstream of TF, the DnaK-DnaJ-GrpE (referred to as KJE) and GroELGroES chaperone systems assist further folding steps to the native state (2, 10). Together these chaperones form a folding network of considerable robustness. Thus, the lack of TF can be compensated by activity of the KJE system, while simultaneous deletion of dnaK and the TF encoding tig gene results in synthetic lethality at Ն30°C and massive protein aggregation (11-13).TF is a three-domain protein of 48 kDa that adopts an unusual extended conformation (Fig. 1A) (5, 14). The N-terminal domain builds the "tail" of the molecule and harbors the "TF-signature motif," which mediates ribosome docking (4). A peptidyl-prolyl cis/trans isomerase (PPIase) domain with structural homology to FK506-binding proteins (9, 16) is located at the other end of ...

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Section: Discussionsupporting

confidence: 62%

Trigger Factor Forms a Protective Shield for Nascent Polypeptides at the Ribosome

Hoffmann

Merz

Rutkowska

et al. 2006

Journal of Biological Chemistry

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“…However, proline isomerization can often be coupled to folding (36,50,51). With increasing TF concentration, the rate of folding was reduced as TF apparently binds and traps folding intermediates of GFPuv, as has been observed for other TF substrates (35,37,50). In this scenario, the limiting step in folding may involve escape from TF to fold in solution, or could reflect the slow folding of GFPuv while bound to TF.…”

Section: Discussionmentioning

confidence: 85%

Trigger Factor Assisted Folding of Green Fluorescent Protein

Xie

Zhou

2007

Biochemistry

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Guanidine induced equilibrium and kinetic folding of a variant of green fluorescent protein (F99S/M153T/V163A, GFPuv) was studied. Using manual mixing and stopped-flow techniques, we combined different probes, including tryptophan fluorescence, chromophore fluorescence and reactivity with DTNB, to trace the spontaneous and TF-assisted folding of guanidine denatured GFPuv. We found that both unfolding and refolding of GFPuv occurred in a stepwise manner and a stable intermediate was populated under equilibrium conditions. The thermodynamic parameters obtained show that the intermediate state of GFPuv is quite compact compared to the denatured state and most of the green fluorescence is retained in this state. By studying GFPuv folding assisted by TF and a number of TF mutants, we found that wild-type TF catalyzes proline isomerization and accelerates the folding rate at low TF concentrations, but retards GFPuv folding and decelerates the folding rate at high TF concentrations. This reflects the two activities of TF, as an enzyme and as a chaperone. A general mechanism of TF assisted protein folding is discussed.

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“…We suggest that the k cat value is related with the dissociation of the substrate protein from the chaperone binding site and its transfer to the prolyl isomerase site. Permanently unfolded substrate proteins dissociate from the chaperone domain of trigger factor with a rate of approximately 8 s Ϫ1 (52). k cat is approximately fivefold lower, probably because dissociation from the chaperone site does not always lead to productive substrate transfer to the prolyl isomerase site.…”

Section: Discussionmentioning

confidence: 99%

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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Dynamic association of trigger factor with protein substrates

Cited by 69 publications

References 36 publications

Trigger Factor Forms a Protective Shield for Nascent Polypeptides at the Ribosome

Trigger Factor Forms a Protective Shield for Nascent Polypeptides at the Ribosome

Trigger Factor Assisted Folding of Green Fluorescent Protein

Chaperone domains convert prolyl isomerases into generic catalysts of protein folding

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