ATPase activity of ClpB_Li and ClpB_Ec.

<p>The rate of ATP hydrolysis was determined at 37°C in the absence of other proteins (basal activity), in the presence of κ-casein (0.1 mg/ml), poly-lysine (0.04 mg/ml) (polyLys), or aggregated G6PDH (2.1 μM) (aggG6PDH). The average values from three independent experiments are shown with the standard deviations.</p

Effect of the <i>clpB</i>_Li gene expression on the growth and survival of <i>E</i>. <i>coli</i> <i>Δ</i><i>clpB</i> mutant under heat shock.

<p>(A) Immunodetection of ClpB_Li with specific antibodies in <i>E</i>. <i>coliΔclpB</i> cells grown at 30°C and after 2h of heat shock at 45°C. An asterisk indicates ClpB_Li. The position of ClpB_Ec (control of heat-inducible expression) was marked by a circle. (B) Growth curves of <i>E</i>. <i>coliΔclpB</i> cells carrying empty pGB2 (control 1), pGB2-ClpB_Ec (control 2) or pGB2-ClpB_Li exposed to a mild heat shock at 45°C for the indicated times. (C) Survival of the same bacterial strains as in (B) after exposure to a severe heat shock at 50°C for the indicated times. The average values from three independent experiments are shown in (B) and (C).</p

Nucleotide-induced oligomerization of ClpB_Li.

<p>Shown are the sedimentation coefficient distributions <i>c</i>(<i>s</i>_20,w) for 1.2 mg/ml ClpB_Li in the absence of nucleotides (A), in the presence of the indicated nucleotide at 2 mM concentration (B-D), and in the low-salt buffer without nucleotides for 3 mg/ml ClpB_Li (E). Sedimentation velocity data presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181118#pone.0181118.g002" target="_blank">Fig 2</a> were analyzed with a continuous sedimentation coefficient distribution <i>c</i>(<i>s</i>) model. The distributions were transformed to standard conditions.</p

Summary of sedimentation coefficients for ClpB_Li.

<p>Summary of sedimentation coefficients for ClpB_Li.</p

Proteolytic sensitivity of ClpB_Li and ClpB_Ec in the absence and presence of nucleotides.

<p>ClpB (1 μM) was incubated at 37°C for the indicated periods with 5 ng of trypsin in the absence or presence of 5 mM nucleotides. The degradation products were resolved by 0.1%SDS-12.5%PAGE and visualized by Coomassie-blue staining. Representative results from three experiments are shown. The positions of standard molecular mass markers (M) (in kDa), PageRuler prestained Protein Ladder (ThermoScientific), are shown on the left.</p

Structural characteristics of ClpB_Li used in this study.

<p>(A) Comparison of the domain organization of ClpB from <i>L</i>. <i>interrogans</i> and <i>E</i>. <i>coli</i>. Bacterial ClpB proteins are composed of the following domains: N-terminal domain (ND), nucleotide binding domain 1 (NBD1), middle coiled-coil domain (MD), and nucleotide binding domain 2 (NBD2). The functions of the domains are indicated at the top. The amino acid residue numbers are shown for each chaperone and the amino acid sequence identity between ClpB_Ec and ClpB_Li is indicated for each domain. (B) CD spectra of ClpB_Li at 20°C (folded form) and 75°C (unfolded form) are shown. The CD signal was expressed as mean molar residue ellipticity (θ). (C) Temperature-induced changes in the CD signal at 222 nm for ClpB_Li.</p

Reactivation of the aggregated substrates in the presence of ClpB_Li and ClpB_Ec.

<p>The reactivation of aggregated enzymes, G6PDH (A) and Fda (B) in the presence of DnaK/DnaJ/GrpE (KJE) from <i>E</i>. <i>coli</i> without ClpB and with ClpB_Ec or ClpB_Li. The native activity of G6PDH or Fda determined before the chemical denaturation or the heat treatment at 55°C, respectively, corresponds to 100%; the fraction of the enzyme activity remaining after the denaturation and also corresponding to the reactivation extent in the absence of chaperones (control) is marked by the broken line. (C) The effect of ClpB_Li and ClpB_Ec on the reactivation of β-galactosidase sequestered into IBs (VP1LAC) isolated from <i>E</i>. <i>coliΔclpB</i> mutant cells. A statistically significant difference in the β-galactosidase activity regain in the absence and presence of ClpB_Li assessed by the paired t-test (using GraphPad Prism software) is indicated as **, p<0.01. The results are presented as the average of three (A, C) or four (B) independent experiments with the standard deviations indicated.</p

Interaction of ClpB_Li with the aggregated G6PDH.

<p>(A) ClpB_Li was incubated with aggregates of G6PDH (Agg) in the presence of 5 mM ATP or ATPγS and without nucleotides. The solutions were passed through a 0.1-μm filter. Subsequently, the fractions retained on the filters were solubilized with an SDS buffer and analyzed by the Coomassie blue-stained 0.1%SDS-10%PAGE gel. A representative result from three independent experiments is shown. (B) Bands corresponding to ClpB_Li were analyzed with Sigma Gel software. Results are presented as the average of three independent experiments with standard deviations indicated. The amount of ClpB_Li detected in the absence of the aggregates is indicated with the broken line.</p

Sedimentation velocity experiments of ClpB_Li.

<p>Radial absorption profiles at 290 nm (•) with the best fits of SEDFIT <i>c</i>(<i>s</i>) model (―) are shown for 1.2 mg/ml (A) and 3 mg/ml ClpB_Li (E) without nucleotides, and for 1.2 mg/ml ClpB_Li with 2 mM nucleotide: ATPγS (B), ADP (C) or AMP-PNP (D). Ultracentrifugation was performed at 50,000 rpm and 20°C. Radial profiles were measured at 4.5-min (A, B, C, E) or 5-min (D) intervals in 50 mM Tris-HCl buffer pH 7.5 containing 20 mM MgCl₂, 2 mM β-mercaptoethanol, 1 mM EDTA, 5% glycerol and 200 mM (A-D) or 30 mM KCl (E). For (A) every second profile is shown for clarity. Bottom panels present the fitting residuals. The time evolution of radial distributions was plotted as colored curves in the order of purple-blue-green-yellow-red.</p

Analysis of the rotor temperature.

<p>(A) Temperature values obtained in different instruments of the spinning rotor, as measured in the iButton at 1,000 rpm after temperature equilibration, while the set point for the console temperature is 20°C (indicated as dotted vertical line). The box-and-whisker plot indicates the central 50% of the data as solid line, with the median displayed as vertical line, and individual circles for data in the upper and lower 25% percentiles. The mean and standard deviation is 19.62°C ± 0.41°C. (B) Correlation between iButton temperature and measured BSA monomer <i>s</i>-values corrected for radial magnification, scan time, scan velocity, but not viscosity (symbols). In addition to the data from the present study as shown in (A) (circles), also shown are measurements from the pilot study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126420#pone.0126420.ref027" target="_blank">27</a>] where the same experiments were carried out on instruments not included in the present study (stars). The dotted line describes the theoretically expected temperature-dependence considering solvent viscosity.</p