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

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
Sequence Assay Result Units