The Experts below are selected from a list of 318 Experts worldwide ranked by ideXlab platform
John Markwell - One of the best experts on this subject based on the ideXlab platform.
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stability of the allergenic soybean kunitz trypsin inhibitor
Biochimica et Biophysica Acta, 2004Co-Authors: Robin Roychaudhuri, Gautam Sarath, Michael G Zeece, John MarkwellAbstract:The soybean Kunitz trypsin inhibitor (SKTI) is a 21.5 kDa allergenic protein that belongs to the family of all antiparallel β-sheet proteins that are highly resistant to thermal and chemical Denaturation. Spectroscopic and biochemical techniques such as circular dichroism (CD), ANS fluorescence and proteolysis were used to study its molecular structure under denaturing conditions such as acid and heat to which these allergens are commonly exposed during food processing. Reduction of native SKTI leads to its complete and rapid proteolysis by pepsin in simulated gastric fluid (SGF). Limited proteolysis with chymotrypsin during renaturation after heating showed that the native structure reforms at around 60 °C reversing the Denaturation. CD spectra revealed that under acid denaturing conditions, SKTI shows major changes in conformation, indicating the possibility of a molten structure. The existence of this intermediate was established by ANS fluorescence studies at different concentrations of HCl. The remarkable stability of SKTI to both thermal and acid Denaturation may be important for its role as a food allergen.
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reversible Denaturation of the soybean kunitz trypsin inhibitor
Archives of Biochemistry and Biophysics, 2003Co-Authors: Robin Roychaudhuri, Gautam Sarath, Michael G Zeece, John MarkwellAbstract:Abstract The soybean Kunitz trypsin inhibitor (SKTI) is a β-sheet protein with unusual stability to chemical and thermal Denaturation. Different spectroscopic criteria were used to follow the thermal Denaturation and renaturation of SKTI. Upon heating to 70 °C, changes in UV difference spectra showed increased absorbance at 292 and 297 nm, attributable to perturbation of aromatic residues. Cooling the protein resulted in restoration of the native spectrum unless reduced with dithiothreitol. Far- and near-UV CD spectra also indicate thermal unfolding involving the core tryptophan and tyrosine residues. Both CD and UV-absorbance data suggest a two-state transition with the midpoint at approximately 65 °C. CD data along with the increased fluorescence intensity of the reporter fluorophore, 1-anilino-8-naphthalenesulfonate with SKTI, between 60 and 70 °C, are consistent with a transition of the native inhibitor to an alternate conformation with a more molten state. Even after heating to 90 °C, subsequent cooling of SKTI resulted in >90% of native trypsin inhibition potential. These results indicate that thermal Denaturation of SKTI is readily reversible to the native form upon cooling and may provide a useful system for future protein folding studies in the class of disordered β-sheet proteins.
Robin Roychaudhuri - One of the best experts on this subject based on the ideXlab platform.
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stability of the allergenic soybean kunitz trypsin inhibitor
Biochimica et Biophysica Acta, 2004Co-Authors: Robin Roychaudhuri, Gautam Sarath, Michael G Zeece, John MarkwellAbstract:The soybean Kunitz trypsin inhibitor (SKTI) is a 21.5 kDa allergenic protein that belongs to the family of all antiparallel β-sheet proteins that are highly resistant to thermal and chemical Denaturation. Spectroscopic and biochemical techniques such as circular dichroism (CD), ANS fluorescence and proteolysis were used to study its molecular structure under denaturing conditions such as acid and heat to which these allergens are commonly exposed during food processing. Reduction of native SKTI leads to its complete and rapid proteolysis by pepsin in simulated gastric fluid (SGF). Limited proteolysis with chymotrypsin during renaturation after heating showed that the native structure reforms at around 60 °C reversing the Denaturation. CD spectra revealed that under acid denaturing conditions, SKTI shows major changes in conformation, indicating the possibility of a molten structure. The existence of this intermediate was established by ANS fluorescence studies at different concentrations of HCl. The remarkable stability of SKTI to both thermal and acid Denaturation may be important for its role as a food allergen.
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reversible Denaturation of the soybean kunitz trypsin inhibitor
Archives of Biochemistry and Biophysics, 2003Co-Authors: Robin Roychaudhuri, Gautam Sarath, Michael G Zeece, John MarkwellAbstract:Abstract The soybean Kunitz trypsin inhibitor (SKTI) is a β-sheet protein with unusual stability to chemical and thermal Denaturation. Different spectroscopic criteria were used to follow the thermal Denaturation and renaturation of SKTI. Upon heating to 70 °C, changes in UV difference spectra showed increased absorbance at 292 and 297 nm, attributable to perturbation of aromatic residues. Cooling the protein resulted in restoration of the native spectrum unless reduced with dithiothreitol. Far- and near-UV CD spectra also indicate thermal unfolding involving the core tryptophan and tyrosine residues. Both CD and UV-absorbance data suggest a two-state transition with the midpoint at approximately 65 °C. CD data along with the increased fluorescence intensity of the reporter fluorophore, 1-anilino-8-naphthalenesulfonate with SKTI, between 60 and 70 °C, are consistent with a transition of the native inhibitor to an alternate conformation with a more molten state. Even after heating to 90 °C, subsequent cooling of SKTI resulted in >90% of native trypsin inhibition potential. These results indicate that thermal Denaturation of SKTI is readily reversible to the native form upon cooling and may provide a useful system for future protein folding studies in the class of disordered β-sheet proteins.
Gautam Sarath - One of the best experts on this subject based on the ideXlab platform.
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stability of the allergenic soybean kunitz trypsin inhibitor
Biochimica et Biophysica Acta, 2004Co-Authors: Robin Roychaudhuri, Gautam Sarath, Michael G Zeece, John MarkwellAbstract:The soybean Kunitz trypsin inhibitor (SKTI) is a 21.5 kDa allergenic protein that belongs to the family of all antiparallel β-sheet proteins that are highly resistant to thermal and chemical Denaturation. Spectroscopic and biochemical techniques such as circular dichroism (CD), ANS fluorescence and proteolysis were used to study its molecular structure under denaturing conditions such as acid and heat to which these allergens are commonly exposed during food processing. Reduction of native SKTI leads to its complete and rapid proteolysis by pepsin in simulated gastric fluid (SGF). Limited proteolysis with chymotrypsin during renaturation after heating showed that the native structure reforms at around 60 °C reversing the Denaturation. CD spectra revealed that under acid denaturing conditions, SKTI shows major changes in conformation, indicating the possibility of a molten structure. The existence of this intermediate was established by ANS fluorescence studies at different concentrations of HCl. The remarkable stability of SKTI to both thermal and acid Denaturation may be important for its role as a food allergen.
-
reversible Denaturation of the soybean kunitz trypsin inhibitor
Archives of Biochemistry and Biophysics, 2003Co-Authors: Robin Roychaudhuri, Gautam Sarath, Michael G Zeece, John MarkwellAbstract:Abstract The soybean Kunitz trypsin inhibitor (SKTI) is a β-sheet protein with unusual stability to chemical and thermal Denaturation. Different spectroscopic criteria were used to follow the thermal Denaturation and renaturation of SKTI. Upon heating to 70 °C, changes in UV difference spectra showed increased absorbance at 292 and 297 nm, attributable to perturbation of aromatic residues. Cooling the protein resulted in restoration of the native spectrum unless reduced with dithiothreitol. Far- and near-UV CD spectra also indicate thermal unfolding involving the core tryptophan and tyrosine residues. Both CD and UV-absorbance data suggest a two-state transition with the midpoint at approximately 65 °C. CD data along with the increased fluorescence intensity of the reporter fluorophore, 1-anilino-8-naphthalenesulfonate with SKTI, between 60 and 70 °C, are consistent with a transition of the native inhibitor to an alternate conformation with a more molten state. Even after heating to 90 °C, subsequent cooling of SKTI resulted in >90% of native trypsin inhibition potential. These results indicate that thermal Denaturation of SKTI is readily reversible to the native form upon cooling and may provide a useful system for future protein folding studies in the class of disordered β-sheet proteins.
Michael G Zeece - One of the best experts on this subject based on the ideXlab platform.
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stability of the allergenic soybean kunitz trypsin inhibitor
Biochimica et Biophysica Acta, 2004Co-Authors: Robin Roychaudhuri, Gautam Sarath, Michael G Zeece, John MarkwellAbstract:The soybean Kunitz trypsin inhibitor (SKTI) is a 21.5 kDa allergenic protein that belongs to the family of all antiparallel β-sheet proteins that are highly resistant to thermal and chemical Denaturation. Spectroscopic and biochemical techniques such as circular dichroism (CD), ANS fluorescence and proteolysis were used to study its molecular structure under denaturing conditions such as acid and heat to which these allergens are commonly exposed during food processing. Reduction of native SKTI leads to its complete and rapid proteolysis by pepsin in simulated gastric fluid (SGF). Limited proteolysis with chymotrypsin during renaturation after heating showed that the native structure reforms at around 60 °C reversing the Denaturation. CD spectra revealed that under acid denaturing conditions, SKTI shows major changes in conformation, indicating the possibility of a molten structure. The existence of this intermediate was established by ANS fluorescence studies at different concentrations of HCl. The remarkable stability of SKTI to both thermal and acid Denaturation may be important for its role as a food allergen.
-
reversible Denaturation of the soybean kunitz trypsin inhibitor
Archives of Biochemistry and Biophysics, 2003Co-Authors: Robin Roychaudhuri, Gautam Sarath, Michael G Zeece, John MarkwellAbstract:Abstract The soybean Kunitz trypsin inhibitor (SKTI) is a β-sheet protein with unusual stability to chemical and thermal Denaturation. Different spectroscopic criteria were used to follow the thermal Denaturation and renaturation of SKTI. Upon heating to 70 °C, changes in UV difference spectra showed increased absorbance at 292 and 297 nm, attributable to perturbation of aromatic residues. Cooling the protein resulted in restoration of the native spectrum unless reduced with dithiothreitol. Far- and near-UV CD spectra also indicate thermal unfolding involving the core tryptophan and tyrosine residues. Both CD and UV-absorbance data suggest a two-state transition with the midpoint at approximately 65 °C. CD data along with the increased fluorescence intensity of the reporter fluorophore, 1-anilino-8-naphthalenesulfonate with SKTI, between 60 and 70 °C, are consistent with a transition of the native inhibitor to an alternate conformation with a more molten state. Even after heating to 90 °C, subsequent cooling of SKTI resulted in >90% of native trypsin inhibition potential. These results indicate that thermal Denaturation of SKTI is readily reversible to the native form upon cooling and may provide a useful system for future protein folding studies in the class of disordered β-sheet proteins.
George M Whitesides - One of the best experts on this subject based on the ideXlab platform.
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influence of fluorocarbon and hydrocarbon acyl groups at the surface of bovine carbonic anhydrase ii on the kinetics of Denaturation by sodium dodecyl sulfate
Journal of Physical Chemistry B, 2011Co-Authors: Andrew K Lee, Katherine A Mirica, George M WhitesidesAbstract:This paper examines the influence of acylation of the Lys-e-NH(3)(+) groups of bovine carbonic anhydrase (BCA, EC 4.2.1.1) to Lys-e-NHCOR (R = -CH(3), -CH(2)CH(3), and -CH(CH(3))(2), -CF(3)) on the rate of Denaturation of this protein in buffer containing sodium dodecyl sulfate (SDS). Analysis of the rates suggested separate effects due to electrostatic charge and hydrophobic interactions. Rates of Denaturation (k(Ac,n)) of each series of acylated derivatives depended on the number of acylations (n). Plots of log k(Ac,n) vs n followed U-shaped curves. Within each series of derivatives, rates of Denaturation decreased as n increased to ∼7; this decrease was compatible with increasingly unfavorable electrostatic interactions between SDS and protein. In this range of n, rates of Denaturation also depended on the choice of the acyl group as n increased to ∼7, in a manner compatible with favorable hydrophobic interactions between SDS and the -NHCOR groups. As n increased in the range 7 < n < 14, however, rates of Denaturation stayed approximately constant; analysis suggested that these rates were compatible with an increasingly important contribution to Denaturation that depended both on the net negative charge of the protein and on the hydrophobicity of the R group. The mechanism of Denaturation thus seems to change with the extent of acylation of the protein. For derivatives with the same net electrostatic charge, rates of Denaturation increased with the acyl group (by a factor of ∼3 for n ∼ 14) in the order CH(3)CONH- < CH(3)CH(2)CONH- < (CH(3))(2)CHCONH- < CF(3)CONH-. These results suggested that the hydrophobicity of CF(3)CONH- is slightly greater (by a factor of <2) than that of RHCONH- with similar surface area.
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increasing the net charge and decreasing the hydrophobicity of bovine carbonic anhydrase decreases the rate of Denaturation with sodium dodecyl sulfate
Biophysical Journal, 2006Co-Authors: Katherine L Gudikse, Irina Gitli, Demetri T Moustakas, George M WhitesidesAbstract:Abstract This study compares the rate of Denaturation with sodium dodecyl sulfate (SDS) of the individual rungs of protein charge ladders generated by acylation of the lysine e − NH 3 + groups of bovine carbonic anhydrase II (BCA). Each acylation decreases the number of positively charged groups, increases the net negative charge, and increases the hydrophobic surface area of BCA. This study reports the kinetics of Denaturation in solutions containing SDS of the protein charge ladders generated with acetic and hexanoic anhydrides; plotting these rates of Denaturation as a function of the number of modifications yields a U-shaped curve. The proteins with an intermediate number of modifications are the most stable to Denaturation by SDS. There are four competing interactions—two resulting from the change in electrostatics and two resulting from the change in exposed hydrophobic surface area—that determine how a modification affects the stability of a rung of a charge ladder of BCA to Denaturation with SDS. A model based on assumptions about how these interactions affect the folded and transition states has been developed and fits the experimental results. Modeling indicates that for each additional acylation, the magnitude of the change in the activation energy of Denaturation (ΔΔ G ‡ ) due to changes in the electrostatics is much larger than the change in ΔΔ G ‡ due to changes in the hydrophobicity, but the intermolecular and intramolecular electrostatic effects are opposite in sign. At the high numbers of acylations, hydrophobic interactions cause the hexanoyl-modified BCA to denature nearly three orders of magnitude more rapidly than the acetyl-modified BCA.