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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

bis-ANS binding to M. leprae HSP18.(A) Fluorescence spectra of bis-ANS bound to HSP18 at different temperatures; (B) Fluorescence spectra of bis-ANS bound to HSP18 in the absence or presence of various concentrations of NaCl at 25°C. Protein concentration in all samples was 0.05 mg/ml and bis-ANS concentration was 10 M. The excitation wavelength was 390 nm. Excitation and emission slit widths were 2.5 and 5 nm, respectively. All the reported spectra were obtained by subtracting the respective control (bis-ANS alone) curves/spectra from the spectra of bis-ANS bound to HSP18.
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pone.0129734.g004: bis-ANS binding to M. leprae HSP18.(A) Fluorescence spectra of bis-ANS bound to HSP18 at different temperatures; (B) Fluorescence spectra of bis-ANS bound to HSP18 in the absence or presence of various concentrations of NaCl at 25°C. Protein concentration in all samples was 0.05 mg/ml and bis-ANS concentration was 10 M. The excitation wavelength was 390 nm. Excitation and emission slit widths were 2.5 and 5 nm, respectively. All the reported spectra were obtained by subtracting the respective control (bis-ANS alone) curves/spectra from the spectra of bis-ANS bound to HSP18.

Mentions: The hallmark of sHSPs is to assemble into large oligomeric assembly of high molecular mass. Often subunits of different sHSPs exchange with each other by dynamic oligomeric dissociation/re-association which is pre-requisite for their chaperone function [16, 24, 37]. Since static oligomeric structure of HSP18 remained unaltered at elevated temperatures (31–43°C), we hypothesised that it is the changes in dynamics of oligomeric structure that caused enhanced chaperone function of HSP18 at these higher temperatures. First, we labelled HSP18 separately with Alexa fluor 350 (A-350) and Alexa flour 488 (A-488) and determined the degree of labeling. We observed that percentage labelling of Alexa fluor 350 and Alexa fluor 488 to HSP18 was 1:1 (M/M) which suggests that all proteins were labelled with these fluorophores. Size exclusion chromatographic experiments revealed that these labelling did not perturb the static oligomeric assembly and exhibited similar oligomeric mass/size compared to that of unlabelled HSP18 (S1 Fig). Then, we monitored the subunit exchange reaction at physiological temperature (37°C) by mixing an equimolar concentration of the donor, Alexa flour 350-labelled HSP18, with the acceptor, Alexa flour 488-labelled HSP18. Due to exchange between subunits of HSP18, we observed marked alteration in the donor and acceptor fluorescence (Fig 3A). Because of FRET, a time dependent decrease in fluorescence intensity at 440 nm for A-350 (labelled to HSP18) was observed with a concomitant time dependent increase in fluorescence intensity at 513 nm for A-488 (labelled to HSP18) which reached to its saturation in 3 hr at 37°C (Fig 4). To find out the rate constant (k) for the subunit exchange, the declining fluorescence intensity of A-350 (Fig 3B) and increasing fluorescence intensity of A-488 (Fig 3C) was fitted to the Eq 3. In both cases, we found that the rate constant (k) for the subunit exchange at 37°C was 0.039 min-1. This value of subunit exchange rate constant for HSP18 is almost comparable with the value of 0.0378 min-1 reported by Bova et al. [46] for the subunit exchange of αA-crystallin at 37°C. In the same paper, they also determined the rate constant for the subunit exchange of HSP27 at 37°C (0.0576 min-1) which is slightly higher compared to that of HSP18 and αA-crystallin. To understand the effect of temperature on the subunit exchange rate of HSP18, rate constant (k) of subunit exchange was also determined at 25, 31 and 43°C (Fig 3D). The subunit exchange rate constants for HSP18 at these three temperatures were 0.018, 0.023 and 0.149 min-1, respectively (Table 2 and Fig 3D). Thus, increasing the temperature from 25°C to 43°C, the magnitude of subunit exchange rate constant for HSP18 was increased almost 8 fold. Moreover, the subunit exchange rate constant was found to markedly increase (~4 fold) for HSP18 when the temperature was raised from 37°C to 43°C. Interestingly, almost similar rise in temperature (37°C to 42°C) also increased the magnitude of subunit exchange rate constant for αA-crystallin by ~4.2 fold [47]. Moreover, we can say that HSP18 possess a dynamic quaternary structure even at 25°C and dynamics of oligomeric dissociation/re-association becomes rapid at elevated temperatures. In several previous studies, investigators demonstrated that the enhancement of the chaperone function of various sHSPs under elevated temperature is mostly associated with increased subunit exchange [16, 24, 47]. Therefore, we concluded that increased subunit exchange of HSP18 subunits at elevated temperature may be one of the basis for the enhancement of its chaperone function at higher 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)

bis-ANS binding to M. leprae HSP18.(A) Fluorescence spectra of bis-ANS bound to HSP18 at different temperatures; (B) Fluorescence spectra of bis-ANS bound to HSP18 in the absence or presence of various concentrations of NaCl at 25°C. Protein concentration in all samples was 0.05 mg/ml and bis-ANS concentration was 10 M. The excitation wavelength was 390 nm. Excitation and emission slit widths were 2.5 and 5 nm, respectively. All the reported spectra were obtained by subtracting the respective control (bis-ANS alone) curves/spectra from the spectra of bis-ANS bound to HSP18.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0129734.g004: bis-ANS binding to M. leprae HSP18.(A) Fluorescence spectra of bis-ANS bound to HSP18 at different temperatures; (B) Fluorescence spectra of bis-ANS bound to HSP18 in the absence or presence of various concentrations of NaCl at 25°C. Protein concentration in all samples was 0.05 mg/ml and bis-ANS concentration was 10 M. The excitation wavelength was 390 nm. Excitation and emission slit widths were 2.5 and 5 nm, respectively. All the reported spectra were obtained by subtracting the respective control (bis-ANS alone) curves/spectra from the spectra of bis-ANS bound to HSP18.
Mentions: The hallmark of sHSPs is to assemble into large oligomeric assembly of high molecular mass. Often subunits of different sHSPs exchange with each other by dynamic oligomeric dissociation/re-association which is pre-requisite for their chaperone function [16, 24, 37]. Since static oligomeric structure of HSP18 remained unaltered at elevated temperatures (31–43°C), we hypothesised that it is the changes in dynamics of oligomeric structure that caused enhanced chaperone function of HSP18 at these higher temperatures. First, we labelled HSP18 separately with Alexa fluor 350 (A-350) and Alexa flour 488 (A-488) and determined the degree of labeling. We observed that percentage labelling of Alexa fluor 350 and Alexa fluor 488 to HSP18 was 1:1 (M/M) which suggests that all proteins were labelled with these fluorophores. Size exclusion chromatographic experiments revealed that these labelling did not perturb the static oligomeric assembly and exhibited similar oligomeric mass/size compared to that of unlabelled HSP18 (S1 Fig). Then, we monitored the subunit exchange reaction at physiological temperature (37°C) by mixing an equimolar concentration of the donor, Alexa flour 350-labelled HSP18, with the acceptor, Alexa flour 488-labelled HSP18. Due to exchange between subunits of HSP18, we observed marked alteration in the donor and acceptor fluorescence (Fig 3A). Because of FRET, a time dependent decrease in fluorescence intensity at 440 nm for A-350 (labelled to HSP18) was observed with a concomitant time dependent increase in fluorescence intensity at 513 nm for A-488 (labelled to HSP18) which reached to its saturation in 3 hr at 37°C (Fig 4). To find out the rate constant (k) for the subunit exchange, the declining fluorescence intensity of A-350 (Fig 3B) and increasing fluorescence intensity of A-488 (Fig 3C) was fitted to the Eq 3. In both cases, we found that the rate constant (k) for the subunit exchange at 37°C was 0.039 min-1. This value of subunit exchange rate constant for HSP18 is almost comparable with the value of 0.0378 min-1 reported by Bova et al. [46] for the subunit exchange of αA-crystallin at 37°C. In the same paper, they also determined the rate constant for the subunit exchange of HSP27 at 37°C (0.0576 min-1) which is slightly higher compared to that of HSP18 and αA-crystallin. To understand the effect of temperature on the subunit exchange rate of HSP18, rate constant (k) of subunit exchange was also determined at 25, 31 and 43°C (Fig 3D). The subunit exchange rate constants for HSP18 at these three temperatures were 0.018, 0.023 and 0.149 min-1, respectively (Table 2 and Fig 3D). Thus, increasing the temperature from 25°C to 43°C, the magnitude of subunit exchange rate constant for HSP18 was increased almost 8 fold. Moreover, the subunit exchange rate constant was found to markedly increase (~4 fold) for HSP18 when the temperature was raised from 37°C to 43°C. Interestingly, almost similar rise in temperature (37°C to 42°C) also increased the magnitude of subunit exchange rate constant for αA-crystallin by ~4.2 fold [47]. Moreover, we can say that HSP18 possess a dynamic quaternary structure even at 25°C and dynamics of oligomeric dissociation/re-association becomes rapid at elevated temperatures. In several previous studies, investigators demonstrated that the enhancement of the chaperone function of various sHSPs under elevated temperature is mostly associated with increased subunit exchange [16, 24, 47]. Therefore, we concluded that increased subunit exchange of HSP18 subunits at elevated temperature may be one of the basis for the enhancement of its chaperone function at higher 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