Secondary structural elements of HSP18 at various temperatures.

<p>Secondary structural elements of HSP18 at various temperatures.</p

Effect of electrostatic interaction on the structural stability of HSP18.

<p>The thermal stability of HSP18 in the absence or presence of 0.5 M NaCl was determined using far-UV CD spectroscopy (panel A) and differential scanning calorimetry (panel C). The experimental data points (mentioned by symbols in panel A and sold lines in panel C) were fitted according to two-state model and the solid lines (in panel A) and dotted lines (in panel C) represent the best fit. <b>(B)</b> The data represented in panel A was fitted to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129734#pone.0129734.e005" target="_blank">Eq 5</a> in order to obtain the values of van’t Hoff enthalpy (∆H_vH) HSP18 in the absence or presence of 0.5 M NaCl.</p

Effect of NaCl on the thermal deactivation prevention ability of <i>M</i>. <i>leprae</i> HSP18.

<p>The MDH enzyme activity was measured in the absence and presence of 30 μM HSP18 pre-incubated in the absence or presence of 0.05–0.5 M NaCl while it was thermally denatured at 43°C. Similar assays were also performed with 30 μM BSA alone or pre-incubated with 0.05–0.5 M NaCl. Data are means ± standard deviation from triplicate determinations. *<i>p</i>< 0.05, **<i>p</i>< 0.005.</p

Effect of NaCl on the chaperone activity of <i>M</i>. <i>leprae</i> HSP18.

<p>DTT-induced aggregation of 0.35 mg/ml insulin at 25°C <b>(panel A)</b> and thermal aggregation of 0.06 mg/ml CS at 43°C <b>(panel C)</b> in the absence or presence of different HSP18 samples. Both insulin and citrate synthase are denoted as client proteins. Trace 1: Client protein (CP) alone; Trace 2: CP +HSP18; Trace 3: CP +0.05 M NaCl; Trace 4: CP + HSP18 + 0.05 M NaCl; Trace 5: CP +0.15 M NaCl; Trace 6: CP + HSP18+0.15 M NaCl; Trace 7: CP +0.5 M NaCl; Trace 8: CP + HSP18+0.5 M NaCl. Each data point is the average of triplicate measurements. The percent protection ability of different HSP18 samples against insulin and CS aggregation are presented in panels B and D, respectively. The insulin: HSP18 ratio was 1:1.2 (w/w) and the CS: HSP18 ratio was 1:1.5 (w/w). Data are means ± standard deviation from triplicate determinations. NS = Not significant, *<i>p</i>< 0.05, **<i>p</i>< 0.005 and ***<i>p</i>< 0.0005.</p

Determination of subunit exchange rate constant of HSP18 subunits at different temperatures.

<p><b>(A)</b> Time-course alterations in the emission spectrum of Alexa fluor-350 and 488 labeled <i>M</i>. <i>leprae</i> HSP18 due to subunit exchange at 37°C. The emission spectra were recorded at different time points after mixing equal amount of Alexa fluor-350 labeled and Alexa fluor-488 labeled HSP18 (1 mg/ml each) at 37°C. The fluorescence spectra were recorded from 400 to 600 nm at 37°C using the excitation wavelength of 346 nm. The slit width of both excitation and emission monochromators was 5 nm each. The scan rate used for this assay was 240 nm/min. <b>(B)</b> Time-dependent decrease in fluorescence intensity at 440 nm for data shown in panel A. <b>(C)</b> Time-dependent increase in fluorescence intensity at 513 nm for data shown in panel A. <b>(D)</b> Temperature-dependent subunit exchange rate of <i>M</i>. <i>leprae</i> HSP18. Subunit exchange between Alexa fluor-350 labeled and Alexa fluor-488 labeled HSP18 was monitored at 25, 31, 37 and 43°C. Symbols in panel B, C and D represent the experimental data points and the solid lines in these pannels represent the best fit of the data according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129734#pone.0129734.e003" target="_blank">Eq 3</a>.</p

Temperature-dependent chaperone-like activities of <i>M</i>. <i>leprae</i> HSP18.

<p><b>(A)</b> DTT-induced aggregation of 0.35 mg/ml insulin (Ins) in the absence and presence 0.35 mg/ml HSP18 at different temperatures (25, 31, 37 and 43°C). Aggregation was initiated by adding 20 mM DTT and scattering at 400 nm was monitored at the respective temperatures. Trace 1: Ins alone at 25°C; Trace 2: Ins + HSP18 at 25°C; Trace 3: Ins alone at 31°C; Trace 4: Ins + HSP18 at 31°C; Trace 5: Ins alone at 37°C; Trace 6: Ins + HSP18 at 37°C; Trace 7: Ins alone at 43°C; Trace 8: Ins + HSP18 at 43°C; <b>(B)</b> Percent protection ability of <i>M</i>. <i>leprae</i> HSP18 against insulin aggregation at different temperatures. Data are means ± the standard deviation from triplicate determinations. **<i>p</i>< 0.005 and ***<i>p</i>< 0.0005.</p

Effect of temperature on the structure of <i>M</i>. <i>leprae</i> HSP18.

<p>Far-UV CD spectra <b>(A)</b> and near-UV spectra <b>(B)</b> of HSP18 at different temperatures (25, 31, 37 and 43°C). The concentrations of the protein samples used in far- and near-UV CD experiments were 0.2 and 0.5 mg/ml, respectively. <b>(C)</b> Tryptophan fluorescence spectra of HSP18 (0.05 mg/ml) were recorded from 310–400 nm at various temperatures (25, 31, 37 and 43°C). An excitation wavelength of 295 nm was used. Both the slit widths for excitation and emission were 5 nm. Data were collected at 0.5 nm wavelength resolution. <b>(D)</b> Intensity particle size distribution spectra of <i>M</i>. <i>leprae</i> HSP18 were recorded at various temperatures (25, 31, 37 and 43°C). Each of these spectra is an average of 48 scans. For each experiment, spectra were recorded after incubating HSP18 at respective temperature for 1 hr.</p

A proposed model which reflects the schematic mechanism for the chaperone function of <i>M</i>. <i>leprae</i> HSP18 at various temperatures.

<p>A proposed model which reflects the schematic mechanism for the chaperone function of <i>M</i>. <i>leprae</i> HSP18 at various temperatures.</p

Effect of NaCl on the oligomeric mass/size of <i>M</i>. <i>leprae</i> HSP18.

<p><b>(A)</b> Gel filtration profile of HSP18 in the absence or presence of 0.05–0.5 M NaCl at 25°C. TSK-GEL G4000SW_XL column (7.8 mm x 30 cm; 5 μm) was first equilibrated with 50 mM phosphate buffer (pH 7.5) with or without 0.05–0.5 M NaCl. Subsequently, 50 μl of HSP18, pre-incubated without or with 0.05–0.5 M NaCl was injected into the column. The flow rate used in this experiment was 0.5 ml/min. The oligomeric mass of different HSP18 samples was estimated using the standard curve (inset). <b>(B)</b> Intensity particle size distribution spectra of <i>M</i>. <i>leprae</i> HSP18 in the absence or presence of 0.05–0.5 M NaCl at 25°C. <b>(C)</b> Intensity particle size distribution spectra of <i>M</i>. <i>leprae</i> HSP18 in the presence of 0.5 M NaCl at different temperatures (25, 31, 37 and 43°C). Each spectrum is an average of 48 scans. HSP18 was incubated at respective temperatures in the absence or presence of 0.05–0.5 M NaCl for 1 hr prior to reading. Protein concentration used was 0.5 mg/ml in 50 mM phosphate buffer, pH 7.5.</p

Effect of temperature on the chaperone activity of <i>M</i>. <i>leprae</i> HSP18 in the presence of NaCl.

<p>The aggregation of insulin B chains (0.35 mg/ml) initiated by the addition of DTT (20 mM) was used to determine the chaperone activity of HSP18 (0.42 mg/ml) at <b>(A)</b> 25°C and <b>(B)</b> 37°C. <b>(C)</b> Percentage protection of insulin (0.35 mg/ml) aggregation by HSP18 in the absence or presence of various NaCl concentrations at two different temperatures. Data are means ± the standard deviation from triplicate determinations. *<i>p</i>< 0.05, **<i>p</i>< 0.005 and ***<i>p</i>< 0.0005.</p