Trigger factor from the psychrophilic bacterium Psychrobacter frigidicola is a monomeric chaperone

Abstract

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.

FIG. 1.

Sequence alignment of trigger factors from P. frigidicola and E. coli. The predicted amino acid sequences of E. coli and P. frigidicola TFs were aligned using CLUSTAL W (34). Invariant residues are indicated on a black background, and similar residues are indicated on a gray background. Predicted secondary structures, determined with PORTER (30), are shown above the TF_Pf and below the TF_Ec sequences: α-helices are drawn as cylinders, β-strands are drawn as arrows, and other elements are drawn as solid lines. The conserved ribosome-binding motif is boxed in dashed lines, and residues conserved in the PPIase catalytic site are indicated by diamonds.

FIG. 2.

PPIase activity of P. frigidicola and E. coli TFs. PPIase activity of TFs against the RCM-RNase T₁ model substrate. RCM-RNase T₁ refolding was monitored by an increase in intrinsic tryptophan fluorescence at 320 nm after excitation at 268 nm. Symbols: •, TF_Pf; ▪, TF_Ec; ×, buffer.

FIG. 3.

Effect of TF concentration on reactivation of GAPDH at 25°C and 15°C. Refolding of denatured GAPDH was monitored by measuring its enzymatic activity 3 h after 50-fold dilution (final concentration 2.5 μM) in the presence of increasing concentrations of TF_Pf (•), TF_Ec (▪), or BSA (×) at 25°C (A) or at 15°C (B).

FIG. 4.

Analysis of quaternary structures of TFs at 25°C and 15°C by cross-linking. Increasing concentrations (indicated by triangles above the lanes) of TF_Pf and TF_Ec were cross-linked using 0.1% glutaraldehyde, precipitated using trichloroacetic acid, and separated by SDS-10% PAGE. (A) Cross-linking at 25°C, with TF concentrations of 0.5, 2.5, 10, 20; and 30 μM; (B) cross-linking at 15°C, with TF concentrations of 0.15, 0.625, 2.5, 10, and 20 μM. Equal amounts of protein were loaded in each lane. The first lane of each TF gradient was not treated with glutaraldehyde. Cross-linked dimers are indicated by stars.

FIG. 5.

Analysis of quaternary structure of TFs at 25°C by fluorescence anisotropy. Alexa Fluor 532-TF (50 nM) was titrated against increasing concentrations of unlabeled TF. The change in anisotropy is plotted against the unlabeled protein concentration, as indicated. Symbols: •, TF_Pf; ▪, TF_Ec.

FIG. 6.

Effect of TFs on the periplasmic export of 2H12 scFv in E. coli. (A) 2H12 scFv was expressed in E. coli BL21(DE3) cells, with TF_Pf and TF_Ec coexpressed from p15aratigPF and p15aratigEC, respectively. Soluble (upper) and insoluble (lower) fractions were analyzed by Western blotting with an anti-His₆ tag antibody. (B) pBADHisB + tig_Pf and + tig_Ec represent E. coli W3110 or E. coli W3110 Δtig cells expressing 2H12 scFv in the presence of pBADHisB control, pBADtigPF, and pBADtigEC plasmids, respectively. Whole-cell extracts were analyzed as described above. Arrows indicate bands of the expected sizes of the processed and unprocessed scFv polypeptides.

FIG. 7.

Effect of increasing concentrations of TFs on cell-free protein synthesis. Various concentrations of TF_Pf and TF_Ec were added to a cell-free protein synthesis system prior to production of the 2H12 scFv recombinant antibody fragment. Activity of the soluble scFv was monitored by ELISA. Soluble and insoluble fractions were separated by centrifugation and analyzed by Western blotting with an anti-M2 FLAG antibody. The results are representative of two independent experiments.