Porcine beta-lactoglobulin chemical unfolding: identification of a non-native alpha-helical intermediate.
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 alpha-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.
D'Alfonso
Laura
L
Dipartimento di Fisica, Universitá degli Studi di Milano-Bicocca, Piazza della Scienza 3, 20126, Milano, Italy.
Collini
Maddalena
M
Ragona
Laura
L
Ugolini
Raffaella
R
Baldini
Giancarlo
G
Molinari
Henriette
H
D000818:Animals; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D007782:Lactoglobulins / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013552:Swine
D007782:Lactoglobulins