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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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Related in: MedlinePlus

Far-UV CD spectra of native (a–c) and refolded (d–f) 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 (d), sucrose (e), and trehalose (f). The native and unfolded enzymes were used as positive and negative controls.
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fig4: Far-UV CD spectra of native (a–c) and refolded (d–f) 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 (d), sucrose (e), and trehalose (f). The native and unfolded enzymes were used as positive and negative controls.

Mentions: The enzyme was mostly in a refolded form as detected by fluorometric experiment; however, the corresponding recovery in TreA activity was less than 56%. Far-UV CD spectra of osmolyte-treated samples were, therefore, measured (Figure 4) and deconvolution of the spectra for secondary structure amount was subsequently performed using CDNN software [35]. Because of the presence of 50.4 mM GdnHCl, the spectra were presented only in the range of 250–205 nm. It was shown that native BlTreA exhibited a strong positive maximum at 192 nm (data not shown) and two negative minima at 208 nm and 222, characteristic of the high α-helix content of the enzyme. The spectral properties of native BlTreA were preserved even after the addition of sugar osmolytes into the enzyme solution (Figure 4). As a control, the GdnHCl-denatured enzyme did not show the typical CD spectra of α-helix. Refolding of GdnHCl-denatured BlTreA in the presence of different concentrations of sugar osmolytes resulted in some recovery of the ellipticity of the protein (Figure 4). The estimates of the secondary content showed that the α-helix and β-strand content of the native enzyme were 45% and 27%, respectively, and those of the partially folded structure were 37–50% and 23–31%, respectively. These results indicate that the intermediate is partially folded in the presence of sugar osmolytes. This situation may be responsible for the incomplete recovery of the TreA activity of the refolded enzyme.


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)

Far-UV CD spectra of native (a–c) and refolded (d–f) 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 (d), sucrose (e), and trehalose (f). 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

fig4: Far-UV CD spectra of native (a–c) and refolded (d–f) 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 (d), sucrose (e), and trehalose (f). The native and unfolded enzymes were used as positive and negative controls.
Mentions: The enzyme was mostly in a refolded form as detected by fluorometric experiment; however, the corresponding recovery in TreA activity was less than 56%. Far-UV CD spectra of osmolyte-treated samples were, therefore, measured (Figure 4) and deconvolution of the spectra for secondary structure amount was subsequently performed using CDNN software [35]. Because of the presence of 50.4 mM GdnHCl, the spectra were presented only in the range of 250–205 nm. It was shown that native BlTreA exhibited a strong positive maximum at 192 nm (data not shown) and two negative minima at 208 nm and 222, characteristic of the high α-helix content of the enzyme. The spectral properties of native BlTreA were preserved even after the addition of sugar osmolytes into the enzyme solution (Figure 4). As a control, the GdnHCl-denatured enzyme did not show the typical CD spectra of α-helix. Refolding of GdnHCl-denatured BlTreA in the presence of different concentrations of sugar osmolytes resulted in some recovery of the ellipticity of the protein (Figure 4). The estimates of the secondary content showed that the α-helix and β-strand content of the native enzyme were 45% and 27%, respectively, and those of the partially folded structure were 37–50% and 23–31%, respectively. These results indicate that the intermediate is partially folded in the presence of sugar osmolytes. This situation may be responsible for the incomplete recovery of the TreA activity of the refolded enzyme.

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