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Beneficial effect of sugar osmolytes on the refolding of guanidine hydrochloride-denatured trehalose-6-phosphate hydrolase from Bacillus licheniformis.

Chen JH, Chi MC, Lin MG, Lin LL, Wang TF - Biomed Res Int (2015)

Bottom Line: These experimental results clearly indicated that sorbitol, sucrose, and trehalose at a concentration of 0.75 M improved the refolding yields of GdnHCl-denatured  BlTreA, probably due to the fact that these sugars favored the formation of tertiary architectures.ANS fluorescence intensity measurements revealed a reduction of exposed hydrophobic surfaces upon the treatment of denatured enzyme with sugar osmolytes.These observations suggest that sugar osmolytes possibly play a chaperone role in the refolding of chemically denatured BlTreA.

View Article: PubMed Central - PubMed

Affiliation: Department of Food Science and Technology, Chia-Nan University of Pharmacy and Science, Tainan City 71710, Taiwan.

ABSTRACT
The influence of three sugar osmolytes on the refolding of guanidine hydrochloride- (GdnHCl-) denatured trehalose-6-phosphate hydrolase of Bacillus licheniformis (BlTreA) was studied by circular dichroism (CD) spectra, fluorescence emission spectra, and the recovery of enzymatic activity. These experimental results clearly indicated that sorbitol, sucrose, and trehalose at a concentration of 0.75 M improved the refolding yields of GdnHCl-denatured  BlTreA, probably due to the fact that these sugars favored the formation of tertiary architectures. Far-UV CD measurements demonstrated the ability of sugar osmolytes to shift the secondary structure of GdnHCl-denatured enzyme towards near-native conformations. ANS fluorescence intensity measurements revealed a reduction of exposed hydrophobic surfaces upon the treatment of denatured enzyme with sugar osmolytes. These observations suggest that sugar osmolytes possibly play a chaperone role in the refolding of chemically denatured BlTreA.

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ANS fluorescence emission spectra of refolded BlTreA in the presence of various concentrations of sugar osmolytes. Refolding occurred by diluting the unfolded enzyme into the standard buffer (50 mM Hepes-NaOH buffer, pH 8.0) in the absence (control) and presence of various concentrations of sugars, including sorbitol (a), sucrose (b), and trehalose (c). The native and unfolded enzymes were used as positive and negative controls.
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fig5: ANS fluorescence emission spectra of refolded BlTreA in the presence of various concentrations of sugar osmolytes. Refolding occurred by diluting the unfolded enzyme into the standard buffer (50 mM Hepes-NaOH buffer, pH 8.0) in the absence (control) and presence of various concentrations of sugars, including sorbitol (a), sucrose (b), and trehalose (c). The native and unfolded enzymes were used as positive and negative controls.

Mentions: The hydrophobic fluorescent dye ANS is widely used to probe the exposure of the hydrophobic region upon protein unfolding. The binding of ANS to hydrophobic regions of proteins results in a profound enhancement of ANS fluorescence intensity and a significant blue-shift of the maximum wavelength. ANS fluorescence intensity measurements of BlTreA refolding in the presence of various amounts of sugar osmolytes were, therefore, employed to investigate the exposure of the hydrophobic groups. As shown in Figure 5, the exposure of hydrophobic surface was markedly reduced upon the addition of sugar osmolytes. These results indicate that BlTreA possess hydrophobic region that is buried in the native state but is exposed under the unfolded state. It also reflects that sugar osmolytes do help the denatured enzyme to refold into the correct native state.


Beneficial effect of sugar osmolytes on the refolding of guanidine hydrochloride-denatured trehalose-6-phosphate hydrolase from Bacillus licheniformis.

Chen JH, Chi MC, Lin MG, Lin LL, Wang TF - Biomed Res Int (2015)

ANS fluorescence emission spectra of refolded BlTreA in the presence of various concentrations of sugar osmolytes. Refolding occurred by diluting the unfolded enzyme into the standard buffer (50 mM Hepes-NaOH buffer, pH 8.0) in the absence (control) and presence of various concentrations of sugars, including sorbitol (a), sucrose (b), and trehalose (c). The native and unfolded enzymes were used as positive and negative controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4309298&req=5

fig5: ANS fluorescence emission spectra of refolded BlTreA in the presence of various concentrations of sugar osmolytes. Refolding occurred by diluting the unfolded enzyme into the standard buffer (50 mM Hepes-NaOH buffer, pH 8.0) in the absence (control) and presence of various concentrations of sugars, including sorbitol (a), sucrose (b), and trehalose (c). The native and unfolded enzymes were used as positive and negative controls.
Mentions: The hydrophobic fluorescent dye ANS is widely used to probe the exposure of the hydrophobic region upon protein unfolding. The binding of ANS to hydrophobic regions of proteins results in a profound enhancement of ANS fluorescence intensity and a significant blue-shift of the maximum wavelength. ANS fluorescence intensity measurements of BlTreA refolding in the presence of various amounts of sugar osmolytes were, therefore, employed to investigate the exposure of the hydrophobic groups. As shown in Figure 5, the exposure of hydrophobic surface was markedly reduced upon the addition of sugar osmolytes. These results indicate that BlTreA possess hydrophobic region that is buried in the native state but is exposed under the unfolded state. It also reflects that sugar osmolytes do help the denatured enzyme to refold into the correct native state.

Bottom Line: These experimental results clearly indicated that sorbitol, sucrose, and trehalose at a concentration of 0.75 M improved the refolding yields of GdnHCl-denatured  BlTreA, probably due to the fact that these sugars favored the formation of tertiary architectures.ANS fluorescence intensity measurements revealed a reduction of exposed hydrophobic surfaces upon the treatment of denatured enzyme with sugar osmolytes.These observations suggest that sugar osmolytes possibly play a chaperone role in the refolding of chemically denatured BlTreA.

View Article: PubMed Central - PubMed

Affiliation: Department of Food Science and Technology, Chia-Nan University of Pharmacy and Science, Tainan City 71710, Taiwan.

ABSTRACT
The influence of three sugar osmolytes on the refolding of guanidine hydrochloride- (GdnHCl-) denatured trehalose-6-phosphate hydrolase of Bacillus licheniformis (BlTreA) was studied by circular dichroism (CD) spectra, fluorescence emission spectra, and the recovery of enzymatic activity. These experimental results clearly indicated that sorbitol, sucrose, and trehalose at a concentration of 0.75 M improved the refolding yields of GdnHCl-denatured  BlTreA, probably due to the fact that these sugars favored the formation of tertiary architectures. Far-UV CD measurements demonstrated the ability of sugar osmolytes to shift the secondary structure of GdnHCl-denatured enzyme towards near-native conformations. ANS fluorescence intensity measurements revealed a reduction of exposed hydrophobic surfaces upon the treatment of denatured enzyme with sugar osmolytes. These observations suggest that sugar osmolytes possibly play a chaperone role in the refolding of chemically denatured BlTreA.

Show MeSH
Related in: MedlinePlus