Porcine beta-lactoglobulin chemical unfolding:: Identification of a non-native α-helical intermediate

被引:13
作者
D'Alfonso, L
Collini, M
Ragona, L
Ugolini, R
Baldini, G
Molinari, H
机构
[1] Univ Milano Bicocca, Dipartimento Fis, I-20126 Milan, Italy
[2] UdR Milano Bicocca, INFM, I-20126 Milan, Italy
[3] CNR, ISMAC, Lab NMR, I-20133 Milan, Italy
[4] Univ Verona, Dipartimento Sci & Tecnol, I-37134 Verona, Italy
关键词
homologous beta-lactoglobulins; unfolding; thermodynamic measurements; non-native intermediates;
D O I
10.1002/prot.20309
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The chemical unfolding behavior of porcine beta-lactoglobulin (PLG) has been followed at pH 2 and 6 in the presence of guanidinium hydrochloride. The PLG unfolding transition, monitored by tryptophan fluorescence, far and near UV circular dichroism and 1D-NMR, can be described by a three-state transition suggesting the presence of at least one intermediate state that appears to display an excess of non-native a-helical structures. The thermodynamic parameters, as determined through a global analysis fitting procedure, give estimates of the free energy differences of the transitions connecting the native, the intermediate and the unfolded state: DeltaG(NI)(0) = 2.8 +/- 0.7 kcal mol(-1) (pH 2) and 4.2 +/- 0.5 kcal mol(-1) (pH 6) and DeltaG(NU)(0) = 7.2 +/- 0.6 kcal mol(-1) (pH 2) and 6.9 +/- 0.6 kcal mol(-1) (pH 6). CD unfolding data of the bovine species (BLG) have been collected here under the same experimental conditions of PLG to allow a careful comparison of the two beta-lactoglobulins. Intermediates with different characteristics have been identified for BLG and PLG, and their nature has been discussed on a structural analysis basis. The thermodynamic data reported here for PLG and BLG and the comparative analysis with data reported for equine beta lactoglobulin, show that homologous beta-barrel proteins' belonging to the same family and displaying high sequence identity (52-64%) populate unfolding intermediates to different extents, even though a common tendency to the formation of non-native alpha-helical intermediates, can be envisaged. The present results provide a prerequisite foundation of knowledge for the design and interpretation of future folding kinetic studies. (C) 2004 Wiley-Liss, Inc.
引用
收藏
页码:70 / 79
页数:10
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