Does beta-lactoglobulin denaturation occur via an intermediate state?


Abstract

The denaturation of beta-lactoglobulin (BLG) in the presence of urea and GuHCl has been investigated at different pH values with various spectroscopic techniques. The equilibrium denaturation free energy values, obtained by linearly extrapolating the data to vanishing denaturant (DeltaG(D)(H2O)), are compared and discussed. The fit of the spectroscopic data monitoring the denaturation of BLG has been approached, at first, with a two-state model that describes the protein transition from the folded state (at each pH and in the absence of denaturant) to the denatured state, but in particular, along the GuHCl denaturation pathway some evidence is found of the presence of an intermediate state. Time-resolved fluorescence experiments performed on the BLG-ANS (1-anilino-8-naphthalenesulfonate) complex help to understand the results. Fluorescence polarization anisotropy (FPA) measurements accompanying the denaturation process show the presence of a fast rotational diffusion of the ANS probe, and the data are interpreted in terms of local fluctuations of a still structured tract of the denatured protein where the probe is bound. Study holds ProTherm entries: 12821, 12822, 12823, 12824, 12825, 12826, 12827, 12828, 12829, 12830, 12831, 12832, 12833, 12834, 12835 Extra Details: free energy; intermediate state; two-state model; local fluctuations

Submission Details

ID: HdbQQSLh

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:44 p.m.

Version: 1

Publication Details
D'Alfonso L;Collini M;Baldini G,Biochemistry (2002) Does beta-lactoglobulin denaturation occur via an intermediate state? PMID:11772032
Additional Information

Study Summary

Number of data points 45
Proteins Beta-lactoglobulin ; Beta-lactoglobulin
Unique complexes 1
Assays/Quantities/Protocols Experimental Assay: Cm pH:8.3, buffers:KHPO4-Na2HPO4/NaOH: ; Experimental Assay: m pH:8.3, buffers:KHPO4-Na2HPO4/NaOH: ; Experimental Assay: dG_H2O pH:8.3, buffers:KHPO4-Na2HPO4/NaOH: ; Experimental Assay: Cm pH:8.3, buffers:KHPO4-Na2HPO4/NaOH: ; Experimental Assay: m pH:8.3, buffers:KHPO4-Na2HPO4/NaOH: ; Experimental Assay: dG_H2O pH:8.3, buffers:KHPO4-Na2HPO4/NaOH: ; Experimental Assay: Cm pH:6.2, buffers:KH2PO4-Na2HPO4: ; Experimental Assay: m pH:6.2, buffers:KH2PO4-Na2HPO4: ; Experimental Assay: dG_H2O pH:6.2, buffers:KH2PO4-Na2HPO4: ; Experimental Assay: Cm pH:6.2, buffers:KH2PO4-Na2HPO4: ; Experimental Assay: m pH:6.2, buffers:KH2PO4-Na2HPO4: ; Experimental Assay: dG_H2O pH:6.2, buffers:KH2PO4-Na2HPO4: ; Experimental Assay: Cm pH:8.3, buffers:KHPO4-Na2HPO4/NaOH: ; Experimental Assay: m pH:8.3, buffers:KHPO4-Na2HPO4/NaOH: ; Experimental Assay: dG_H2O pH:8.3, buffers:KHPO4-Na2HPO4/NaOH: ; Experimental Assay: Cm pH:8.3, buffers:KHPO4-Na2HPO4/NaOH: ; Experimental Assay: m pH:8.3, buffers:KHPO4-Na2HPO4/NaOH: ; Experimental Assay: dG_H2O pH:8.3, buffers:KHPO4-Na2HPO4/NaOH: ; Experimental Assay: Cm pH:6.2, buffers:KH2PO4-Na2HPO4: ; Experimental Assay: m pH:6.2, buffers:KH2PO4-Na2HPO4: ; Experimental Assay: dG_H2O pH:6.2, buffers:KH2PO4-Na2HPO4: ; Experimental Assay: Cm pH:6.2, buffers:KH2PO4-Na2HPO4: ; Experimental Assay: m pH:6.2, buffers:KH2PO4-Na2HPO4: ; Experimental Assay: dG_H2O pH:6.2, buffers:KH2PO4-Na2HPO4: ; Experimental Assay: Cm pH:2.3, buffers:H3PO4, NaOH: ; Experimental Assay: m pH:2.3, buffers:H3PO4, NaOH: ; Experimental Assay: dG_H2O pH:2.3, buffers:H3PO4, NaOH: ; Experimental Assay: Cm pH:2.3, buffers:H3PO4, NaOH: ; Experimental Assay: m pH:2.3, buffers:H3PO4, NaOH: ; Experimental Assay: dG_H2O pH:2.3, buffers:H3PO4, NaOH:
Libraries 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
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)
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
4OMW 2014-01-27T00:00:00+0000 2.3 Crystal structure of goat beta-lactoglobulin (orthorhombic form)
4OMX 2014-01-27T00:00:00+0000 2.3 Crystal structure of goat beta-lactoglobulin (trigonal form)
4TLJ 2014-05-30T00:00:00+0000 1.17 Ultra-high resolution crystal structure of caprine Beta-lactoglobulin
4Y0S 2015-02-06T00:00:00+0000 1.9 Goat beta-lactoglobulin complex with pramocaine (GLG-PRM)
7LWC 2021-02-28T00:00:00+0000 3.0 Goat beta-lactoglobulin mutant Q59A
1B0O 1998-11-11T00:00:00+0000 2.5 BOVINE BETA-LACTOGLOBULIN COMPLEXED WITH PALMITATE, LATTICE Z

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