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Porcine beta‐lactoglobulin chemical unfolding: Identification of a non‐native α‐helical intermediate
Author(s) -
D'Alfonso Laura,
Collini Maddalena,
Ragona Laura,
Ugolini Raffaella,
Baldini Giancarlo,
Molinari Henriette
Publication year - 2004
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.20309
Subject(s) - chemistry , circular dichroism , native state , guanidine , crystallography , equilibrium unfolding , intermediate state , tryptophan , molten globule , homologous series , stereochemistry , amino acid , biochemistry , philosophy , theology
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 α‐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: ΔG NI 0= 2.8 ± 0.7 kcal mol −1 (pH 2) and 4.2 ± 0.5 kcal mol −1 (pH 6) and ΔG 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. Proteins 2005. © 2004 Wiley‐Liss, Inc.