Identification of a potential hydrophobic peptide binding site in the C‐terminal arm of trigger factor

Shi, Yi; Fan, Dongjie; Li, Shuxin; Zhang, Hongjie; Perrett, Sarah; Zhou, Jun‐Mei

doi:10.1110/ps.062623707

Cited by 17 publications

(8 citation statements)

References 48 publications

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“…This first characterization of a TF from a psychrophilic species has revealed striking properties of TF Pf . In combination with other recent studies in the field (18,25,32,37), it significantly advances our molecular understanding of the functioning of TF. Given in particular that a specific mechanistic role has been proposed for dimeric TF Ec in posttranslational protein folding (20), the present results highlight that dimerization may not be a universal feature among bacteria and that the physiological relevance of dimer formation remains to be fully understood.…”

Section: Resultsmentioning

confidence: 83%

Trigger Factor from the Psychrophilic Bacterium Psychrobacter frigidicola Is a Monomeric Chaperone

et al. 2009

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In eubacteria, trigger factor (TF) is the first chaperone to interact with newly synthesized polypeptides and assist their folding as they emerge from the ribosome. We report the first characterization of a TF from a psychrophilic organism. TF from Psychrobacter frigidicola (TF Pf ) was cloned, produced in Escherichia coli, and purified. Strikingly, cross-linking and fluorescence anisotropy analyses revealed it to exist in solution as a monomer, unlike the well-characterized, dimeric E. coli TF (TF Ec ). Moreover, TF Pf did not exhibit the downturn in reactivation of unfolded GAPDH (glyceraldehyde-3-phosphate dehydrogenase) that is observed with its E. coli counterpart, even at high TF/GAPDH molar ratios and revealed dramatically reduced retardation of membrane translocation by a model recombinant protein compared to the E. coli chaperone. TF Pf was also significantly more effective than TF Ec at increasing the yield of soluble and functional recombinant protein in a cell-free protein synthesis system, indicating that it is not dependent on downstream systems for its chaperoning activity. We propose that TF Pf differs from TF Ec in its quaternary structure and chaperone activity, and we discuss the potential significance of these differences in its native environment.The folding of cytosolic proteins is coordinated by three chaperone systems in eubacteria: trigger factor (TF), DnaK and its cofactors DnaJ and GrpE, and GroEL, together with its cofactor GroES. As TF alone interacts with the ribosomes, it is the first chaperone to bind newly synthesized polypeptides and assist their folding, whereupon it passes them to downstream chaperone systems (12). It has recently been shown that, for multidomain proteins, TF and the DnaK system cooperate to slow down folding and cause a shift to a posttranslational folding mode (24). TF also plays an important role in the regulation of protein translocation due to its position at the ribosome exit tunnel (1, 24).TF is highly abundant in E. coli (up to 40 M), with most TF molecules existing in an equilibrium between its monomeric and dimeric states in the cytosol (28). The high cytosolic concentration of TF has recently been linked to a distinct functional role of the chaperone in maintaining newly translated polypeptides in a folding-competent state in the E. coli cytoplasm (35). A distinct antichaperone activity has also been described for TF, however, whereby substoichiometric concentrations of TF lead to increased polypeptide aggregation, whereas high TF/polypeptide ratios can also delay folding as the chaperone maintains polypeptides in a non-native state without promoting their complete refolding (9).E. coli TF (TF Ec ) is composed of an N-terminal ribosomebinding tail (domain I), an internal domain with peptidyl-prolyl cis/trans isomerase (PPIase) activity (domain II), and a Cterminal domain III that is involved in its chaperone activity (26). The importance of the PPIase domain remains unclear as TF binds non-native nascent polypeptides lacking proline residues (29), w...

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Section: Resultsmentioning

confidence: 83%

Trigger Factor from the Psychrophilic Bacterium Psychrobacter frigidicola Is a Monomeric Chaperone

et al. 2009

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“…Bis-ANS fluorescence increases upon binding hydrophobic regions of proteins, and is therefore widely employed to probe this property 31,32. Based on this literature, we reasoned that Bis-ANS may reveal differences between in Aβ oligomers and fibrils.…”

Section: Resultsmentioning

confidence: 99%

A chemical screening approach reveals that indole fluorescence is quenched by pre-fibrillar but not fibrillar amyloid-β

Reinke¹,

Seh²,

Gestwicki³

2009

Bioorganic & Medicinal Chemistry Letters

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Aggregated amyloid-β (Aβ) peptide is implicated in the pathology of Alzheimer's disease. In vitro and in vivo, these aggregates are found in a variety of morphologies, including globular oligomers and linear fibrils, which possess distinct biological activities. However, known chemical probes, including the dyes thioflavin T and Congo Red, appear to lack selectivity for specific amyloid structures. To identify molecules that might differentiate between these architectures, we employed a fluorescence-based interaction assay to screen a collection of 68 known Aβ ligands against preformed oligomers and fibrils. In these studies, we found that the fluorescence of five indole-based compounds was selectively quenched (~15%) in the presence of oligomers, but remained unchanged after addition of fibrils. These results suggest that indoles might be complementary to existing chemical probes for studying amyloid formation in vitro.

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“…Based on photocross-linking, both arms were found to be adjacent to the nascent chain during translation (17,24). Experiments using 1-anilino-8-naphthalene-sulfonate (ANS) as a hydrophobicity sensor showed cross-linking of the probe to residues 391-392 in arm 2 (25). Modeling of TF bound to the archaeal 50 S ribosomal subunit suggested that the C-domain provides a shielded space over the opening of the ribosomal exit tunnel for the folding of small protein domains (18).…”

mentioning

confidence: 99%

Versatility of Trigger Factor Interactions with Ribosome-Nascent Chain Complexes

Lakshmipathy

Gupta²,

Pinkert

et al. 2010

Journal of Biological Chemistry

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Trigger factor (TF) is the first molecular chaperone that interacts with nascent chains emerging from bacterial ribosomes. TF is a modular protein, consisting of an N-terminal ribosome binding domain, a PPIase domain, and a C-terminal domain, all of which participate in polypeptide binding. To directly monitor the interactions of TF with nascent polypeptide chains, TF variants were site-specifically labeled with an environmentally sensitive NBD fluorophore. We found a marked increase in TF-NBD fluorescence during translation of firefly luciferase (Luc) chains, which expose substantial regions of hydrophobicity, but not with nascent chains lacking extensive hydrophobic segments. TF remained associated with Luc nascent chains for 111 ؎ 7 s, much longer than it remained bound to the ribosomes (t1 ⁄ 2 ϳ 10 -14 s). Thus, multiple TF molecules can bind per nascent chain during translation. The Escherichia coli cytosolic proteome was classified into predicted weak and strong interactors for TF, based on the occurrence of continuous hydrophobic segments in the primary sequence. The residence time of TF on the nascent chain generally correlated with the presence of hydrophobic regions and the capacity of nascent chains to bury hydrophobicity. Interestingly, TF bound the signal sequence of a secretory protein, pOmpA, but not the hydrophobic signal anchor sequence of the inner membrane protein FtsQ. On the other hand, proteins lacking linear hydrophobic segments also recruited TF, suggesting that TF can recognize hydrophobic surface features discontinuous in sequence. Moreover, TF retained significant affinity for the folded domain of the positively charged, ribosomal protein S7, indicative of an alternative mode of TF action. Thus, unlike other chaperones, TF appears to employ multiple mechanisms to interact with a wide range of substrate proteins.

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Identification of a potential hydrophobic peptide binding site in the C‐terminal arm of trigger factor

Cited by 17 publications

References 48 publications

Trigger Factor from the Psychrophilic Bacterium Psychrobacter frigidicola Is a Monomeric Chaperone

Trigger Factor from the Psychrophilic Bacterium Psychrobacter frigidicola Is a Monomeric Chaperone

A chemical screening approach reveals that indole fluorescence is quenched by pre-fibrillar but not fibrillar amyloid-β

Versatility of Trigger Factor Interactions with Ribosome-Nascent Chain Complexes

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