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Role of Subunit Exchange and Electrostatic Interactions on the Chaperone Activity of Mycobacterium leprae HSP18.

Nandi SK, Panda AK, Chakraborty A, Sinha Ray S, Biswas A - PLoS ONE (2015)

Bottom Line: At elevated temperatures, weakening of interactions between HSP18 and stressed client proteins in the presence of NaCl results in greater reduction of its chaperone function.The oligomeric size, rate of subunit exchange and structural stability of HSP18 were also found to decrease when electrostatic interactions were weakened.These results clearly indicated that subunit exchange and electrostatic interactions play a major role in the chaperone function of HSP18.

View Article: PubMed Central - PubMed

Affiliation: School of Basic Sciences, Indian Institute of Technology Bhubaneswar, Bhubaneswar, India.

ABSTRACT
Mycobacterium leprae HSP18, a major immunodominant antigen of M. leprae pathogen, is a small heat shock protein. Previously, we reported that HSP18 is a molecular chaperone that prevents aggregation of different chemically and thermally stressed client proteins and assists refolding of denatured enzyme at normal temperature. We also demonstrated that it can efficiently prevent the thermal killing of E. coli at higher temperature. However, molecular mechanism behind the chaperone function of HSP18 is still unclear. Therefore, we studied the structure and chaperone function of HSP18 at normal temperature (25°C) as well as at higher temperatures (31-43°C). Our study revealed that the chaperone function of HSP18 is enhanced significantly with increasing temperature. Far- and near-UV CD experiments suggested that its secondary and tertiary structure remain intact in this temperature range (25-43°C). Besides, temperature has no effect on the static oligomeric size of this protein. Subunit exchange study demonstrated that subunits of HSP18 exchange at 25°C with a rate constant of 0.018 min(-1). Both rate of subunit exchange and chaperone activity of HSP18 is found to increase with rise in temperature. However, the surface hydrophobicity of HSP18 decreases markedly upon heating and has no correlation with its chaperone function in this temperature range. Furthermore, we observed that HSP18 exhibits diminished chaperone function in the presence of NaCl at 25°C. At elevated temperatures, weakening of interactions between HSP18 and stressed client proteins in the presence of NaCl results in greater reduction of its chaperone function. The oligomeric size, rate of subunit exchange and structural stability of HSP18 were also found to decrease when electrostatic interactions were weakened. These results clearly indicated that subunit exchange and electrostatic interactions play a major role in the chaperone function of HSP18.

No MeSH data available.


Related in: MedlinePlus

Effect of temperature on the structure of M. leprae HSP18.Far-UV CD spectra (A) and near-UV spectra (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. (C) 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. (D) Intensity particle size distribution spectra of M. leprae 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.
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pone.0129734.g002: Effect of temperature on the structure of M. leprae HSP18.Far-UV CD spectra (A) and near-UV spectra (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. (C) 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. (D) Intensity particle size distribution spectra of M. leprae 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.

Mentions: In order to understand whether the enhancement in the chaperone activity of HSP18 at elevated temperature is accompanied by any alteration in the proteins’ conformations, secondary, tertiary and quaternary structure/conformation was studied at different temperatures (25, 31, 37 and 43°C). Far-UV CD spectra of HSP18 at these four temperatures are shown in Fig 2A, which clearly indicate that temperature has no effect on the secondary structure of HSP18. Data represented in Table 1 clearly indicated that HSP18 is a major β-sheet protein at 25°C. However, with increasing temperature, no change in the secondary structural elements of HSP18 was observed (Table 1). These data further confirmed that secondary structure of HSP18 was not altered upon the temperature rise from 25°C to 43°C. In addition, the effect of temperature/heat on the tertiary structure of HSP18 was studied using near-UV CD experiment (Fig 2B). The signal between 250–270 nm region arose from the five phenylalanine residues in HSP18 (Fig 2B). The signal beyond 270 nm corresponds to the single tyrosine and tryptophan residue in HSP18 (Fig 2B). When the near-UV CD spectra of HSP18 was recorded at elevated temperatures i.e. 31, 37 and 43°C, the spectral characteristics remained unaltered compared to that recorded at 25°C (Fig 2B). These data indicated that microenvironment of aromatic amino acid residues in HSP18 was not perturbed due to increase in temperature. The intrinsic tryptophan fluorescence spectra of HSP18 at different temperatures (25, 31, 37 and 43°C) agreed with the near-UV CD results (Fig 2C). No alteration in λmax as well as intensity of the intrinsic tryptophan fluorescence spectra was observed at higher temperature which further confirmed that temperature did not perturb the microenvironment of the single tryptophan residue (W33) of HSP18.


Role of Subunit Exchange and Electrostatic Interactions on the Chaperone Activity of Mycobacterium leprae HSP18.

Nandi SK, Panda AK, Chakraborty A, Sinha Ray S, Biswas A - PLoS ONE (2015)

Effect of temperature on the structure of M. leprae HSP18.Far-UV CD spectra (A) and near-UV spectra (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. (C) 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. (D) Intensity particle size distribution spectra of M. leprae 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.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4476693&req=5

pone.0129734.g002: Effect of temperature on the structure of M. leprae HSP18.Far-UV CD spectra (A) and near-UV spectra (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. (C) 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. (D) Intensity particle size distribution spectra of M. leprae 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.
Mentions: In order to understand whether the enhancement in the chaperone activity of HSP18 at elevated temperature is accompanied by any alteration in the proteins’ conformations, secondary, tertiary and quaternary structure/conformation was studied at different temperatures (25, 31, 37 and 43°C). Far-UV CD spectra of HSP18 at these four temperatures are shown in Fig 2A, which clearly indicate that temperature has no effect on the secondary structure of HSP18. Data represented in Table 1 clearly indicated that HSP18 is a major β-sheet protein at 25°C. However, with increasing temperature, no change in the secondary structural elements of HSP18 was observed (Table 1). These data further confirmed that secondary structure of HSP18 was not altered upon the temperature rise from 25°C to 43°C. In addition, the effect of temperature/heat on the tertiary structure of HSP18 was studied using near-UV CD experiment (Fig 2B). The signal between 250–270 nm region arose from the five phenylalanine residues in HSP18 (Fig 2B). The signal beyond 270 nm corresponds to the single tyrosine and tryptophan residue in HSP18 (Fig 2B). When the near-UV CD spectra of HSP18 was recorded at elevated temperatures i.e. 31, 37 and 43°C, the spectral characteristics remained unaltered compared to that recorded at 25°C (Fig 2B). These data indicated that microenvironment of aromatic amino acid residues in HSP18 was not perturbed due to increase in temperature. The intrinsic tryptophan fluorescence spectra of HSP18 at different temperatures (25, 31, 37 and 43°C) agreed with the near-UV CD results (Fig 2C). No alteration in λmax as well as intensity of the intrinsic tryptophan fluorescence spectra was observed at higher temperature which further confirmed that temperature did not perturb the microenvironment of the single tryptophan residue (W33) of HSP18.

Bottom Line: At elevated temperatures, weakening of interactions between HSP18 and stressed client proteins in the presence of NaCl results in greater reduction of its chaperone function.The oligomeric size, rate of subunit exchange and structural stability of HSP18 were also found to decrease when electrostatic interactions were weakened.These results clearly indicated that subunit exchange and electrostatic interactions play a major role in the chaperone function of HSP18.

View Article: PubMed Central - PubMed

Affiliation: School of Basic Sciences, Indian Institute of Technology Bhubaneswar, Bhubaneswar, India.

ABSTRACT
Mycobacterium leprae HSP18, a major immunodominant antigen of M. leprae pathogen, is a small heat shock protein. Previously, we reported that HSP18 is a molecular chaperone that prevents aggregation of different chemically and thermally stressed client proteins and assists refolding of denatured enzyme at normal temperature. We also demonstrated that it can efficiently prevent the thermal killing of E. coli at higher temperature. However, molecular mechanism behind the chaperone function of HSP18 is still unclear. Therefore, we studied the structure and chaperone function of HSP18 at normal temperature (25°C) as well as at higher temperatures (31-43°C). Our study revealed that the chaperone function of HSP18 is enhanced significantly with increasing temperature. Far- and near-UV CD experiments suggested that its secondary and tertiary structure remain intact in this temperature range (25-43°C). Besides, temperature has no effect on the static oligomeric size of this protein. Subunit exchange study demonstrated that subunits of HSP18 exchange at 25°C with a rate constant of 0.018 min(-1). Both rate of subunit exchange and chaperone activity of HSP18 is found to increase with rise in temperature. However, the surface hydrophobicity of HSP18 decreases markedly upon heating and has no correlation with its chaperone function in this temperature range. Furthermore, we observed that HSP18 exhibits diminished chaperone function in the presence of NaCl at 25°C. At elevated temperatures, weakening of interactions between HSP18 and stressed client proteins in the presence of NaCl results in greater reduction of its chaperone function. The oligomeric size, rate of subunit exchange and structural stability of HSP18 were also found to decrease when electrostatic interactions were weakened. These results clearly indicated that subunit exchange and electrostatic interactions play a major role in the chaperone function of HSP18.

No MeSH data available.


Related in: MedlinePlus