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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

Temperature-dependent chaperone-like activities of M. leprae HSP18.(A) 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) Percent protection ability of M. leprae HSP18 against insulin aggregation at different temperatures. Data are means ± the standard deviation from triplicate determinations. **p< 0.005 and ***p< 0.0005.
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pone.0129734.g001: Temperature-dependent chaperone-like activities of M. leprae HSP18.(A) 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) Percent protection ability of M. leprae HSP18 against insulin aggregation at different temperatures. Data are means ± the standard deviation from triplicate determinations. **p< 0.005 and ***p< 0.0005.

Mentions: To accomplish this, we first investigated the chaperone activity of M. leprae HSP18 at different temperatures. As the normal body temperature of human being, the host of pathogen M. leprae is ~37°C, the temperature range that has been selected for this study is 37 ± 6°C i.e. 31, 37 and 43°C. Apart from these three temperatures, room temperature (25°C) was also selected. Effect of heat/temperature on the chaperone function of HSP18 was assessed by monitoring the ability of HSP18 to prevent aggregation of insulin B chain. It was co-incubated with insulin at different temperatures (25, 31, 37 and 43°C) for 1 hr and DTT induced insulin aggregation assay was performed at the respective temperatures. At a ratio of 1:1 (w/w) of HSP18 to insulin, 24.6% protection against insulin aggregation was observed at 25°C (Fig 1A, trace 2 and 1B). Surprisingly, at the same chaperone to client protein ratio, the protection was increased to ~43% at 31°C (Fig 1A, trace 4 and 1B). When the temperature was further increased to 37°C, a further increase in the protection against insulin aggregation was observed (~68%) (Fig 1A, trace 6 and 1B). HSP18 protected substantially/almost the insulin aggregation (90%) at 43°C (Fig 1A, trace 8 and 1B). Therefore, it is quite evident from here that the temperature has profound influence on the aggregation prevention ability of HSP18. The chaperone function of HSP18 increases with increasing temperature.


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)

Temperature-dependent chaperone-like activities of M. leprae HSP18.(A) 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) Percent protection ability of M. leprae HSP18 against insulin aggregation at different temperatures. Data are means ± the standard deviation from triplicate determinations. **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.g001: Temperature-dependent chaperone-like activities of M. leprae HSP18.(A) 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) Percent protection ability of M. leprae HSP18 against insulin aggregation at different temperatures. Data are means ± the standard deviation from triplicate determinations. **p< 0.005 and ***p< 0.0005.
Mentions: To accomplish this, we first investigated the chaperone activity of M. leprae HSP18 at different temperatures. As the normal body temperature of human being, the host of pathogen M. leprae is ~37°C, the temperature range that has been selected for this study is 37 ± 6°C i.e. 31, 37 and 43°C. Apart from these three temperatures, room temperature (25°C) was also selected. Effect of heat/temperature on the chaperone function of HSP18 was assessed by monitoring the ability of HSP18 to prevent aggregation of insulin B chain. It was co-incubated with insulin at different temperatures (25, 31, 37 and 43°C) for 1 hr and DTT induced insulin aggregation assay was performed at the respective temperatures. At a ratio of 1:1 (w/w) of HSP18 to insulin, 24.6% protection against insulin aggregation was observed at 25°C (Fig 1A, trace 2 and 1B). Surprisingly, at the same chaperone to client protein ratio, the protection was increased to ~43% at 31°C (Fig 1A, trace 4 and 1B). When the temperature was further increased to 37°C, a further increase in the protection against insulin aggregation was observed (~68%) (Fig 1A, trace 6 and 1B). HSP18 protected substantially/almost the insulin aggregation (90%) at 43°C (Fig 1A, trace 8 and 1B). Therefore, it is quite evident from here that the temperature has profound influence on the aggregation prevention ability of HSP18. The chaperone function of HSP18 increases with increasing temperature.

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