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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 chaperone activity of M. leprae HSP18 in the presence of NaCl.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 (A) 25°C and (B) 37°C. (C) 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. *p< 0.05, **p< 0.005 and ***p< 0.0005.
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pone.0129734.g007: Effect of temperature on the chaperone activity of M. leprae HSP18 in the presence of NaCl.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 (A) 25°C and (B) 37°C. (C) 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. *p< 0.05, **p< 0.005 and ***p< 0.0005.

Mentions: We also checked the chaperone activity of HSP18 at physiological temperature (37°C) in the absence and presence of different NaCl concentrations (0.15 and 0.5 M) using DTT induced insulin aggregation assay. In the presence of 0.15 M NaCl, the chaperone function of HSP18 was found to decrease by ~17.5% at 37°C (Fig 7B and 7C), while the same was found to decrease by ~11.6% at 25°C (Fig 7A and 7C). The more dramatic effect was observed in the presence 0.5 M NaCl. At this NaCl concentration, we noted that the chaperone function of HSP18 was lowered by ~21.6% at 25°C (Fig 7A and 7C). However, at the same salt concentration, the protection ability of HSP18 was reduced significantly (~45%) at 37°C (Fig 7B and 7C). We also found the greater reduction in the chaperone activity of HSP18 in the presence of 0.15 M and 0.5 M NaCl at other two higher temperatures (31°C and 43°C) (data not shown). Together, our data revealed that the interactions between HSP18 and chemically stressed client protein were weakened greatly in the presence of NaCl at higher temperature, which is the cause for reduction in the chaperone function of HSP18. Possibly, electrostatic interactions govern the recognition and binding of aggregation prone substrate proteins by HSP18 at normal as well as elevated temperatures.


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 chaperone activity of M. leprae HSP18 in the presence of NaCl.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 (A) 25°C and (B) 37°C. (C) 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. *p< 0.05, **p< 0.005 and ***p< 0.0005.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0129734.g007: Effect of temperature on the chaperone activity of M. leprae HSP18 in the presence of NaCl.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 (A) 25°C and (B) 37°C. (C) 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. *p< 0.05, **p< 0.005 and ***p< 0.0005.
Mentions: We also checked the chaperone activity of HSP18 at physiological temperature (37°C) in the absence and presence of different NaCl concentrations (0.15 and 0.5 M) using DTT induced insulin aggregation assay. In the presence of 0.15 M NaCl, the chaperone function of HSP18 was found to decrease by ~17.5% at 37°C (Fig 7B and 7C), while the same was found to decrease by ~11.6% at 25°C (Fig 7A and 7C). The more dramatic effect was observed in the presence 0.5 M NaCl. At this NaCl concentration, we noted that the chaperone function of HSP18 was lowered by ~21.6% at 25°C (Fig 7A and 7C). However, at the same salt concentration, the protection ability of HSP18 was reduced significantly (~45%) at 37°C (Fig 7B and 7C). We also found the greater reduction in the chaperone activity of HSP18 in the presence of 0.15 M and 0.5 M NaCl at other two higher temperatures (31°C and 43°C) (data not shown). Together, our data revealed that the interactions between HSP18 and chemically stressed client protein were weakened greatly in the presence of NaCl at higher temperature, which is the cause for reduction in the chaperone function of HSP18. Possibly, electrostatic interactions govern the recognition and binding of aggregation prone substrate proteins by HSP18 at normal as well as elevated temperatures.

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