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

Study Summary

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 VEVTPIMTELDTQKVAGTWHTVAMAVSDVSLLDAKSSPLKAYVEGLKPTPEGDLEILLQKRENDKCAQEVLLAKKTDIPAVFKINALDENQLFLLDTDYDSHLLLCMENSASPEHSLVCQSLARTLEVDDQIREKFEDALKTLSVPMRILPAQLEEQCRV ; Mutations for sequence LIVTQTMKGLDIQKVAGTWYSLAMAASDISLLDAQSAPLRVYVEELKPTPEGDLEILLQKWENGECAQKKIIAEKTKIPAVFKIDALNENKVLVLDTDYKKYLLFCMENSAEPEQSLACQCLVRTPEVDDEALEKFDKALKALPMHIRLSFNPTQLEEQCHI

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1EXS 2000-05-04T00:00:00+0000 2.39 STRUCTURE OF PORCINE BETA-LACTOGLOBULIN
4NLJ 2013-11-14T00:00:00+0000 1.4 Crystal structure of sheep beta-lactoglobulin (space group P1)
6T44 2019-10-12T00:00:00+0000 2.0 Ovine lactoglobulin complex with decanol
4CK4 2013-12-27T00:00:00+0000 1.12 Ovine beta-Lactoglobulin at Atomic Resolution
4NLI 2013-11-14T00:00:00+0000 1.9 Crystal structure of sheep beta-lactoglobulin (space group P3121)
4OMX 2014-01-27T00:00:00+0000 2.3 Crystal structure of goat beta-lactoglobulin (trigonal form)
4Y0S 2015-02-06T00:00:00+0000 1.9 Goat beta-lactoglobulin complex with pramocaine (GLG-PRM)
4OMW 2014-01-27T00:00:00+0000 2.3 Crystal structure of goat beta-lactoglobulin (orthorhombic form)
7LWC 2021-02-28T00:00:00+0000 3.0 Goat beta-lactoglobulin mutant Q59A
4TLJ 2014-05-30T00:00:00+0000 1.17 Ultra-high resolution crystal structure of caprine Beta-lactoglobulin

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
95.7 Beta-lactoglobulin P67975 LACB_OVIMU
95.7 Beta-lactoglobulin P67976 LACB_SHEEP
96.3 Beta-lactoglobulin P02756 LACB_CAPHI
98.8 Beta-lactoglobulin P02755 LACB_BUBBU
100.0 Beta-lactoglobulin P02754 LACB_BOVIN
100.0 Beta-lactoglobulin-1A/1C P04119 LACB_PIG