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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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Intrinsic 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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fig3: Intrinsic 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 reversibility of unfolding was studied using various parameters including the exposure of tryptophan residues, loss of secondary structure, and the exposure of hydrophobic surface. Tryptophan fluorescence emission spectrum of native BlTreA was characterized by a peak centered at 331.4 nm. The fluorescence markedly changes when the protein was in the unfolded state, with a shift in the emission maximum to 355.6 nm [30]. When GdnHCl-denatured BlTreA was diluted with the refolding buffer containing various amounts of sorbitol, sucrose, and trehalose, there was a rapid return of the fluorescence wavelength maximum to 336.2 nm (Figure 3). The blue shift of the maximum of fluorescence emission from the unfolded state suggests that most of the Trp residues have recovered the environment as they have in the 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)

Intrinsic 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

fig3: Intrinsic 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 reversibility of unfolding was studied using various parameters including the exposure of tryptophan residues, loss of secondary structure, and the exposure of hydrophobic surface. Tryptophan fluorescence emission spectrum of native BlTreA was characterized by a peak centered at 331.4 nm. The fluorescence markedly changes when the protein was in the unfolded state, with a shift in the emission maximum to 355.6 nm [30]. When GdnHCl-denatured BlTreA was diluted with the refolding buffer containing various amounts of sorbitol, sucrose, and trehalose, there was a rapid return of the fluorescence wavelength maximum to 336.2 nm (Figure 3). The blue shift of the maximum of fluorescence emission from the unfolded state suggests that most of the Trp residues have recovered the environment as they have in the 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