Porcine beta-lactoglobulin chemical unfolding: identification of a non-native alpha-helical intermediate.


Abstract

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. Study holds ProTherm entries: 19276, 19277, 19278, 19279, 19280, 19281, 19282 Extra Details: Native to Intermediate homologous beta-lactoglobulins; unfolding; thermodynamic measurements; non-native intermediates

Submission Details

ID: AHCTaVzH3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:51 p.m.

Version: 1

Publication Details
D'Alfonso L;Collini M;Ragona L;Ugolini R;Baldini G;Molinari H,Proteins (2005) Porcine beta-lactoglobulin chemical unfolding: identification of a non-native alpha-helical intermediate. PMID:15526300
Additional Information

Number of data points 21
Proteins Beta-lactoglobulin ; Beta-lactoglobulin ; Beta-lactoglobulin-1A/1C ; Beta-lactoglobulin-1A/1C
Unique complexes 2
Assays/Quantities/Protocols Experimental Assay: Cm pH:6.0, prot_conc:40-60 microM ; Experimental Assay: m pH:6.0, prot_conc:40-60 microM ; Experimental Assay: dG_H2O pH:6.0, prot_conc:40-60 microM ; Experimental Assay: Cm pH:2.0, prot_conc:40-60 microM ; Experimental Assay: m pH:2.0, prot_conc:40-60 microM ; Experimental Assay: dG_H2O pH:2.0, prot_conc:40-60 microM ; Experimental Assay: Cm pH:6.0, prot_conc:1 microM ; Experimental Assay: m pH:6.0, prot_conc:1 microM ; Experimental Assay: dG_H2O pH:6.0, prot_conc:1 microM ; Experimental Assay: Cm pH:6.0, prot_conc:10 microM ; Experimental Assay: m pH:6.0, prot_conc:10 microM ; Experimental Assay: dG_H2O pH:6.0, prot_conc:10 microM ; Experimental Assay: Cm pH:2.0, prot_conc:1 microM ; Experimental Assay: m pH:2.0, prot_conc:1 microM ; Experimental Assay: dG_H2O pH:2.0, prot_conc:1 microM ; Experimental Assay: Cm prot_conc:10 microM, pH:2.0 ; Experimental Assay: m prot_conc:10 microM, pH:2.0 ; Experimental Assay: dG_H2O prot_conc:10 microM, pH:2.0
Libraries Mutations for sequence LIVTQTMKGLDIQKVAGTWYSLAMAASDISLLDAQSAPLRVYVEELKPTPEGDLEILLQKWENGECAQKKIIAEKTKIPAVFKIDALNENKVLVLDTDYKKYLLFCMENSAEPEQSLACQCLVRTPEVDDEALEKFDKALKALPMHIRLSFNPTQLEEQCHI ; Mutations for sequence VEVTPIMTELDTQKVAGTWHTVAMAVSDVSLLDAKSSPLKAYVEGLKPTPEGDLEILLQKRENDKCAQEVLLAKKTDIPAVFKINALDENQLFLLDTDYDSHLLLCMENSASPEHSLVCQSLARTLEVDDQIREKFEDALKTLSVPMRILPAQLEEQCRV
Sequence Assay Result Units