INTRODUCTION Polyols are known to improve the stability of protein folding, but the exact mechanism of their stabilizing action remains debated. Beta-lactoglobulin (BLG) is a relatively small protein with a well know denaturation behavior. BLG is a major food allergen, and also has been proposed as a possible drug carrier. Denaturation of BLG by chemical and physical agents is known to occur through a number of steps, each affecting separate regions of the protein. In this study, BLG was used to assess the effects of high concentrations of sucrose, sorbitol, glycerol, or trehalose on the unfolding of various structural regions in BLG. METHODS Stabilizing effects towards physical and chemical denaturation were assessed by near-UV circular dichroism, and by evaluating the reactivity of Cys121, hidden in the native state of BLG. Surface hydrophobic interaction were detected by using suitable non-covalent fluorescent probes. Effects of polyols on the dissociation equilibrium of the BLG native dimer were assessed by estimating a diffusion coefficient through NMR spectroscopy. RESULTS Polyols in solution shift the Native/Denatured equilibrium towards the native state. However, individual polyols have region-specific stabilizing effects, suggesting a unique mechanism of action for each of them. The effect of polyols on the overall denaturation of BLG (detected by CD) show a close correlation with the ability of each polyol to improve surface hydrophobic interactions (i.e. hydrophobic interaction within the excluded region). Soluble BLG aggregates were observed only at the highest concentrations of polyols. CONCLUSIONS The different nature and extent of the stabilizing effects reported here are discussed in terms of the preferential exclusion theory (polyols interacting only with the solvent and not with the protein), and in terms of the effects of individual polyols on water activity, in order to clarify structure-specific aspects of the stabilization effects.
Different polyols selectively stabilize specific structural regions in beta-lactoglobulin / A. Barbiroli, D. Fessas, E. Ragg, S. Iametti, S. Renzetti, F. Bonomi. ((Intervento presentato al 58. convegno National Meeting of the Italian Society of Biochemistry and Molecular Biology tenutosi a Urbino nel 2015.
Different polyols selectively stabilize specific structural regions in beta-lactoglobulin
A. BarbiroliPrimo
;D. FessasSecondo
;E. Ragg;S. Iametti;F. BonomiUltimo
2015
Abstract
INTRODUCTION Polyols are known to improve the stability of protein folding, but the exact mechanism of their stabilizing action remains debated. Beta-lactoglobulin (BLG) is a relatively small protein with a well know denaturation behavior. BLG is a major food allergen, and also has been proposed as a possible drug carrier. Denaturation of BLG by chemical and physical agents is known to occur through a number of steps, each affecting separate regions of the protein. In this study, BLG was used to assess the effects of high concentrations of sucrose, sorbitol, glycerol, or trehalose on the unfolding of various structural regions in BLG. METHODS Stabilizing effects towards physical and chemical denaturation were assessed by near-UV circular dichroism, and by evaluating the reactivity of Cys121, hidden in the native state of BLG. Surface hydrophobic interaction were detected by using suitable non-covalent fluorescent probes. Effects of polyols on the dissociation equilibrium of the BLG native dimer were assessed by estimating a diffusion coefficient through NMR spectroscopy. RESULTS Polyols in solution shift the Native/Denatured equilibrium towards the native state. However, individual polyols have region-specific stabilizing effects, suggesting a unique mechanism of action for each of them. The effect of polyols on the overall denaturation of BLG (detected by CD) show a close correlation with the ability of each polyol to improve surface hydrophobic interactions (i.e. hydrophobic interaction within the excluded region). Soluble BLG aggregates were observed only at the highest concentrations of polyols. CONCLUSIONS The different nature and extent of the stabilizing effects reported here are discussed in terms of the preferential exclusion theory (polyols interacting only with the solvent and not with the protein), and in terms of the effects of individual polyols on water activity, in order to clarify structure-specific aspects of the stabilization effects.
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