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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 electrostatic interaction on the structural stability of HSP18.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) The data represented in panel A was fitted to Eq 5 in order to obtain the values of van’t Hoff enthalpy (∆HvH) HSP18 in the absence or presence of 0.5 M NaCl.
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pone.0129734.g008: Effect of electrostatic interaction on the structural stability of HSP18.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) The data represented in panel A was fitted to Eq 5 in order to obtain the values of van’t Hoff enthalpy (∆HvH) HSP18 in the absence or presence of 0.5 M NaCl.

Mentions: Once it was concluded that electrostatic interactions are important for the chaperone function of M. leprae HSP18, we were keen to know the molecular basis for the decreased chaperone function of HSP18 when the electrostatic forces were weakened by addition of NaCl. Electrostatic interactions are noncovalent bonding interactions, either attractive or repulsive in nature. It includes coulombic interactions and more sequence specific interactions like ion pairing or hydrogen bonding [52]. Electrostatic interaction is known to play key role in protein stability and function [53, 54]. It has long been implicated in maintaining thermal stability of thermophilic proteins [55, 56]. Valle et al. reported that Ca2+ ion induced decreased thermal stability of α-crystallin caused mainly due to electrostatic interaction between Ca2+/α-crystallin which eventually led to its decreased chaperone functionality [57]. So, the structural stability of HSP18 in the absence and presence of 0.5 M NaCl was compared against thermal stress using far-UV CD measurements. The change in the ellipticity magnitude at 222 nm was monitored over a temperature range from 25°C to 80°C. Then, the fraction unfolded (αU) for both systems were calculated using the following equation:αU=θF−θTθF−θUEq 4where θF represents ellipticity value measured at 25°C for the native protein, θt represents ellipticity value measured at any temperature and θU represents ellipticity value measured at 80°C for the unfolded protein. As shown in Fig 8A, the thermal denaturation profiles of both proteins (HSP18 in the absence and presence of 0.5 M NaCl) were sigmoidal in nature. Although formation of an intermediate state cannot be negated, a two state transition model exhibited a significantly good fitting. Sigmoidal analysis of far-UV CD profiles at 222 nm demonstrated that HSP18 in absence of any NaCl underwent thermal unfolding with a midpoint transition or melting temperature (Tm) of 60.9°C (Table 3). In the presence of 0.5 M NaCl, the Tm value shifted to 54°C (Fig 8A and Table 3). The decrease in Tm value (~6.9°C) clearly suggested that the presence of NaCl significantly reduced the structural stability of HSP18. As thermal denaturation profiles of HSP18 in the absence and presence of 0.5 M NaCl gave a significantly good fit with the two state transition model, the change in enthalpy (∆HvH) of this thermal transition was evaluated using van’t Hoff equation:lnKeq(Folded→Unfolded)=−ΔHvHRT+ΔSREq 5where Keq(folded→unfolded) = αU/(1 - αU). The value of van’t Hoff enthalpy (ΔHvH) associated with the thermal transition of HSP18 (in absence of NaCl) was ~122.5 kJ/mol, which decreased to 92.2 kJ/mol for HSP18 in the presence of 0.5 M NaCl (Fig 8B and Table 3) which further confirmed that weakening of electrostatic interactions lowered the structural stability of HSP18 under thermal stress. The reversibility of this unfolding process of HSP18 in the absence or presence of 0.5 M NaCl was also checked by reheating the protein sample immediately after cooling at 25°C from the previous scan. It is worthwhile to mention that the Tm and ΔHvH values so obtained from the reheating process were similar to that of during first heating. Therefore, it was inferred that the unfolding of HSP18 in the absence and presence of 0.5 M NaCl is reversible in nature.


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 electrostatic interaction on the structural stability of HSP18.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) The data represented in panel A was fitted to Eq 5 in order to obtain the values of van’t Hoff enthalpy (∆HvH) HSP18 in the absence or presence of 0.5 M NaCl.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0129734.g008: Effect of electrostatic interaction on the structural stability of HSP18.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) The data represented in panel A was fitted to Eq 5 in order to obtain the values of van’t Hoff enthalpy (∆HvH) HSP18 in the absence or presence of 0.5 M NaCl.
Mentions: Once it was concluded that electrostatic interactions are important for the chaperone function of M. leprae HSP18, we were keen to know the molecular basis for the decreased chaperone function of HSP18 when the electrostatic forces were weakened by addition of NaCl. Electrostatic interactions are noncovalent bonding interactions, either attractive or repulsive in nature. It includes coulombic interactions and more sequence specific interactions like ion pairing or hydrogen bonding [52]. Electrostatic interaction is known to play key role in protein stability and function [53, 54]. It has long been implicated in maintaining thermal stability of thermophilic proteins [55, 56]. Valle et al. reported that Ca2+ ion induced decreased thermal stability of α-crystallin caused mainly due to electrostatic interaction between Ca2+/α-crystallin which eventually led to its decreased chaperone functionality [57]. So, the structural stability of HSP18 in the absence and presence of 0.5 M NaCl was compared against thermal stress using far-UV CD measurements. The change in the ellipticity magnitude at 222 nm was monitored over a temperature range from 25°C to 80°C. Then, the fraction unfolded (αU) for both systems were calculated using the following equation:αU=θF−θTθF−θUEq 4where θF represents ellipticity value measured at 25°C for the native protein, θt represents ellipticity value measured at any temperature and θU represents ellipticity value measured at 80°C for the unfolded protein. As shown in Fig 8A, the thermal denaturation profiles of both proteins (HSP18 in the absence and presence of 0.5 M NaCl) were sigmoidal in nature. Although formation of an intermediate state cannot be negated, a two state transition model exhibited a significantly good fitting. Sigmoidal analysis of far-UV CD profiles at 222 nm demonstrated that HSP18 in absence of any NaCl underwent thermal unfolding with a midpoint transition or melting temperature (Tm) of 60.9°C (Table 3). In the presence of 0.5 M NaCl, the Tm value shifted to 54°C (Fig 8A and Table 3). The decrease in Tm value (~6.9°C) clearly suggested that the presence of NaCl significantly reduced the structural stability of HSP18. As thermal denaturation profiles of HSP18 in the absence and presence of 0.5 M NaCl gave a significantly good fit with the two state transition model, the change in enthalpy (∆HvH) of this thermal transition was evaluated using van’t Hoff equation:lnKeq(Folded→Unfolded)=−ΔHvHRT+ΔSREq 5where Keq(folded→unfolded) = αU/(1 - αU). The value of van’t Hoff enthalpy (ΔHvH) associated with the thermal transition of HSP18 (in absence of NaCl) was ~122.5 kJ/mol, which decreased to 92.2 kJ/mol for HSP18 in the presence of 0.5 M NaCl (Fig 8B and Table 3) which further confirmed that weakening of electrostatic interactions lowered the structural stability of HSP18 under thermal stress. The reversibility of this unfolding process of HSP18 in the absence or presence of 0.5 M NaCl was also checked by reheating the protein sample immediately after cooling at 25°C from the previous scan. It is worthwhile to mention that the Tm and ΔHvH values so obtained from the reheating process were similar to that of during first heating. Therefore, it was inferred that the unfolding of HSP18 in the absence and presence of 0.5 M NaCl is reversible in nature.

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