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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 NaCl on the chaperone activity of M. leprae HSP18.DTT-induced aggregation of 0.35 mg/ml insulin at 25°C (panel A) and thermal aggregation of 0.06 mg/ml CS at 43°C (panel C) 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, *p< 0.05, **p< 0.005 and ***p< 0.0005.
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pone.0129734.g005: Effect of NaCl on the chaperone activity of M. leprae HSP18.DTT-induced aggregation of 0.35 mg/ml insulin at 25°C (panel A) and thermal aggregation of 0.06 mg/ml CS at 43°C (panel C) 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, *p< 0.05, **p< 0.005 and ***p< 0.0005.

Mentions: Failing to find a direct correlation between chaperone function and surface hydrophobicity in this temperature range prompted us to investigate some other forces which may be responsible for the enhanced chaperone function of HSP18 at higher temperature. In some of the sHSPs (α-crystallin and M. tuberculosis HSP16.3) it has been reported that, besides the hydrophobic interactions, the chaperone function of these sHSPs are also dependent on the electrostatic/ionic interactions [27, 28]. Charge-charge/electrostatic interactions are key to the substrate binding properties of these sHSPs. Therefore, to understand whether electrostatic interactions play a role in the chaperone function of M. leprae HSP18, we investigated the chaperone activity of HSP18 in the absence or presence of NaCl (0.05–0.5M) using DTT induced aggregation of insulin at 25°C. The effect of different concentrations of NaCl on the aggregation of insulin in the absence or presence of HSP18 is shown in Fig 5A. At 1:1.2 (w/w) ratio of insulin to HSP18, 49% protection against aggregation was observed in the absence of NaCl (Fig 5A, trace 2 and 5B). In the presence of 0.05 M NaCl, protection was reduced to ~42% (Fig 5A, trace 4 and 5B). As the NaCl concentration was increased from 0.05 M to 0.5 M, the protection ability of HSP18 was found to decrease further (~27%) (Fig 5A, trace 8 and 5B). The insulin aggregation assay was also carried out at 25°C by varying the ratio between HSP18 and insulin and 0.15 M NaCl. In both cases, the chaperone activity of HSP18 was found to decrease in the presence of 0.15 M NaCl (S2 Fig). To check, whether the effect is substrate specific or not, we examined the aggregation prevention ability of HSP18 in the absence and presence of NaCl (0.05–0.5 M) using another client protein i.e. citrate synthase (CS). Usually, CS tends to aggregate upon heating at 43°C. At 1:1.5 (w/w) ratio of CS to HSP18, 54% protection was observed in the absence of NaCl (Fig 5C, trace 2 and 5D). However, in the presence of 0.05 M NaCl, the protection ability was reduced to ~36% (Fig 5C, trace 4 and 5D). Upon further increase in NaCl concentrations (0.15 and 0.5 M), the protection ability was further reduced (27% and 21%, respectively) (Fig 5C, traces 6 & 8 and 5D). Thus from both the aggregation assays it was inferred that HSP18 exhibits reduced chaperone activity in the presence of NaCl. Besides aggregation prevention ability, HSP18 can also prevent the thermal inactivation of enzyme [malate dehydrogenase (MDH)] [33]. To understand whether the weakening of electrostatic interactions impair this ability of HSP18, the thermal deactivation of MDH was carried out without or with different HSP18 protein samples (pre-incubated without or with 0.05–0.5 M NaCl concentrations) (Fig 6). MDH alone could retain only 39% of its activity. However, in the presence of 30 μM HSP18, it could retain ~63% enzyme activity (Fig 6). When HSP18 was pre-incubated with 0.05–0.5 M NaCl and then subjected to MDH thermal deactivation assay, the loss of MDH activity was increased and it could only retain ~57% (at 0.05 M NaCl) to ~46% (at 0.5 M NaCl) enzyme activity (Fig 6). So as to check the specificity, parallel experiments were also carried out where HSP18 was replaced with BSA (Fig 6), but, BSA failed to exhibit thermal deactivation prevention ability both in the absence or presence of 0.05–0.5 M NaCl (Fig 6). Appropriate controls were also done to check the enzymatic activity of MDH in the presence of various concentrations of NaCl without HSP18. Thus it was inferred that the thermal deactivation prevention ability of HSP18 was reduced in the presence of NaCl. Besides, the effect of NaCl on the surface hydrophobicity of HSP18 was also monitored using bis-ANS experiment. Interestingly, with varying concentrations of NaCl ranging from 0–0.5 M, the fluorescence intensity of bis-ANS bound to HSP18 was found to increase by ~19% at 25°C (Fig 4B), suggesting over the fact that surface hydrophobicity of HSP18 was enhanced in the presence of NaCl. However, the chaperone function of HSP18 was reduced in the presence of NaCl at the same temperature. Therefore, it can be concluded from these evidences that electrostatic interactions play a crucial role for proper execution of chaperone function by M. leprae HSP18 at 25°C.


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 NaCl on the chaperone activity of M. leprae HSP18.DTT-induced aggregation of 0.35 mg/ml insulin at 25°C (panel A) and thermal aggregation of 0.06 mg/ml CS at 43°C (panel C) 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, *p< 0.05, **p< 0.005 and ***p< 0.0005.
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pone.0129734.g005: Effect of NaCl on the chaperone activity of M. leprae HSP18.DTT-induced aggregation of 0.35 mg/ml insulin at 25°C (panel A) and thermal aggregation of 0.06 mg/ml CS at 43°C (panel C) 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, *p< 0.05, **p< 0.005 and ***p< 0.0005.
Mentions: Failing to find a direct correlation between chaperone function and surface hydrophobicity in this temperature range prompted us to investigate some other forces which may be responsible for the enhanced chaperone function of HSP18 at higher temperature. In some of the sHSPs (α-crystallin and M. tuberculosis HSP16.3) it has been reported that, besides the hydrophobic interactions, the chaperone function of these sHSPs are also dependent on the electrostatic/ionic interactions [27, 28]. Charge-charge/electrostatic interactions are key to the substrate binding properties of these sHSPs. Therefore, to understand whether electrostatic interactions play a role in the chaperone function of M. leprae HSP18, we investigated the chaperone activity of HSP18 in the absence or presence of NaCl (0.05–0.5M) using DTT induced aggregation of insulin at 25°C. The effect of different concentrations of NaCl on the aggregation of insulin in the absence or presence of HSP18 is shown in Fig 5A. At 1:1.2 (w/w) ratio of insulin to HSP18, 49% protection against aggregation was observed in the absence of NaCl (Fig 5A, trace 2 and 5B). In the presence of 0.05 M NaCl, protection was reduced to ~42% (Fig 5A, trace 4 and 5B). As the NaCl concentration was increased from 0.05 M to 0.5 M, the protection ability of HSP18 was found to decrease further (~27%) (Fig 5A, trace 8 and 5B). The insulin aggregation assay was also carried out at 25°C by varying the ratio between HSP18 and insulin and 0.15 M NaCl. In both cases, the chaperone activity of HSP18 was found to decrease in the presence of 0.15 M NaCl (S2 Fig). To check, whether the effect is substrate specific or not, we examined the aggregation prevention ability of HSP18 in the absence and presence of NaCl (0.05–0.5 M) using another client protein i.e. citrate synthase (CS). Usually, CS tends to aggregate upon heating at 43°C. At 1:1.5 (w/w) ratio of CS to HSP18, 54% protection was observed in the absence of NaCl (Fig 5C, trace 2 and 5D). However, in the presence of 0.05 M NaCl, the protection ability was reduced to ~36% (Fig 5C, trace 4 and 5D). Upon further increase in NaCl concentrations (0.15 and 0.5 M), the protection ability was further reduced (27% and 21%, respectively) (Fig 5C, traces 6 & 8 and 5D). Thus from both the aggregation assays it was inferred that HSP18 exhibits reduced chaperone activity in the presence of NaCl. Besides aggregation prevention ability, HSP18 can also prevent the thermal inactivation of enzyme [malate dehydrogenase (MDH)] [33]. To understand whether the weakening of electrostatic interactions impair this ability of HSP18, the thermal deactivation of MDH was carried out without or with different HSP18 protein samples (pre-incubated without or with 0.05–0.5 M NaCl concentrations) (Fig 6). MDH alone could retain only 39% of its activity. However, in the presence of 30 μM HSP18, it could retain ~63% enzyme activity (Fig 6). When HSP18 was pre-incubated with 0.05–0.5 M NaCl and then subjected to MDH thermal deactivation assay, the loss of MDH activity was increased and it could only retain ~57% (at 0.05 M NaCl) to ~46% (at 0.5 M NaCl) enzyme activity (Fig 6). So as to check the specificity, parallel experiments were also carried out where HSP18 was replaced with BSA (Fig 6), but, BSA failed to exhibit thermal deactivation prevention ability both in the absence or presence of 0.05–0.5 M NaCl (Fig 6). Appropriate controls were also done to check the enzymatic activity of MDH in the presence of various concentrations of NaCl without HSP18. Thus it was inferred that the thermal deactivation prevention ability of HSP18 was reduced in the presence of NaCl. Besides, the effect of NaCl on the surface hydrophobicity of HSP18 was also monitored using bis-ANS experiment. Interestingly, with varying concentrations of NaCl ranging from 0–0.5 M, the fluorescence intensity of bis-ANS bound to HSP18 was found to increase by ~19% at 25°C (Fig 4B), suggesting over the fact that surface hydrophobicity of HSP18 was enhanced in the presence of NaCl. However, the chaperone function of HSP18 was reduced in the presence of NaCl at the same temperature. Therefore, it can be concluded from these evidences that electrostatic interactions play a crucial role for proper execution of chaperone function by M. leprae HSP18 at 25°C.

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