Dataset Information

Protein self-association in solution: the bovine beta -lactoglobulin dimer and octamer.

ABSTRACT: We have used proton magnetic relaxation dispersion (MRD) to study the self-association of bovine beta-lactoglobulin variant A (BLG-A) as a function of temperature at pH 4.7 (dimer-octamer equilibrium) and as a function of NaCl concentration at pH 2.5 (monomer-dimer equilibrium). The MRD method identifies coexisting oligomers from their rotational correlation times and determines their relative populations from the associated dispersion amplitudes. From MRD-derived correlation times and hydrodynamic model calculations, we confirm that BLG-A dimers associate to octamers below room temperature. The tendency for BLG-A dimers to assemble into octamers is found to be considerably weaker than in previous light scattering studies in the presence of buffer salt. At pH 2.5, the MRD data are consistent with an essentially complete transition from monomers in the absence of salt to dimers in 1 M NaCl. Because of an interfering relaxation dispersion from nanosecond water exchange, we cannot determine the oligomer populations at intermediate salt concentrations. This nanosecond dispersion may reflect intersite exchange of water molecules trapped inside the large binding cavity of BLG-A.

SUBMITTER: Gottschalk M

PROVIDER: S-EPMC2366967 | biostudies-literature | 2003 Nov

REPOSITORIES: biostudies-literature

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Protein self-association in solution: the bovine beta -lactoglobulin dimer and octamer.

Gottschalk Michael M Nilsson Hanna H Roos Helena H Halle Bertil B

Protein science : a publication of the Protein Society 20031101 11

We have used proton magnetic relaxation dispersion (MRD) to study the self-association of bovine beta-lactoglobulin variant A (BLG-A) as a function of temperature at pH 4.7 (dimer-octamer equilibrium) and as a function of NaCl concentration at pH 2.5 (monomer-dimer equilibrium). The MRD method identifies coexisting oligomers from their rotational correlation times and determines their relative populations from the associated dispersion amplitudes. From MRD-derived correlation times and hydrodyna ...[more]

PMID: 14573854

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Project description:Although bovine beta-lactoglobulin assumes a monomeric native structure at pH 3 in the absence of salt, the addition of salts stabilizes the dimer. Thermodynamics of the monomer-dimer equilibrium dependent on the salt concentration were studied by sedimentation equilibrium. The addition of NaCl, KCl, or guanidine hydrochloride below 1 M stabilized the dimer in a similar manner. On the other hand, NaClO(4) was more effective than other salts by about 20-fold, suggesting that anion binding is responsible for the salt-induced dimer formation, as observed for acid-unfolded proteins. The addition of guanidine hydrochloride at 5 M dissociated the dimer into monomers because of the denaturation of protein structure. In the presence of either NaCl or NaClO(4), the dimerization constant decreased with an increase in temperature, indicating that the enthalpy change (DeltaH(D)) of dimer formation is negative. The heat effect of the dimer formation was directly measured with an isothermal titration calorimeter by titrating the monomeric beta-lactoglobulin at pH 3.0 with NaClO(4). The net heat effects after subtraction of the heat of salt dilution, corresponding to DeltaH(D), were negative, and were consistent with those obtained by the sedimentation equilibrium. From the dependence of dimerization constant on temperature measured by sedimentation equilibrium, we estimated the DeltaH(D) value at 20 degrees C and the heat capacity change (DeltaC(p)) of dimer formation. In both NaCl and NaClO(4), the obtained DeltaC(p) value was negative, indicating the dominant role of burial of the hydrophobic surfaces upon dimer formation. The observed DeltaC(p) values were consistent with the calculated value from the X-ray dimeric structure using a method of accessible surface area. These results indicated that monomer-dimer equilibrium of beta-lactoglobulin at pH 3 is determined by a subtle balance of hydrophobic and electrostatic effects, which are modulated by the addition of salts or by changes in temperature.

Project description:Bovine beta-lactoglobulin A assumes a dimeric native conformation at neutral pH, while the conformation at pH 2 is monomeric but still native. Beta-lactoglobulin A has a free thiol at Cys121, which is buried between the beta-barrel and the C-terminal major alpha-helix. This thiol group was specifically reacted with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in the presence of 1.0 M Gdn-HCI at pH 7.5, producing a modified beta-lactoglobulin (TNB-bIg) containing a mixed disulfide bond with 5-thio-2-nitrobenzoic acid (TNB). The conformation and stability of TNB-bIg were studied by circular dichroism (CD), tryptophan fluorescence, analytical ultracentrifugation, and one-dimensional 1H-NMR. The CD spectra of TNB-bIg indicated disordering of the native secondary structure at pH 7.5, whereas a slight increase in the alpha-helical content was observed at pH 2.0. The tryptophan fluorescence of TNB-bIg was significantly quenched compared with that of the intact protein, probably by the energy transfer to TNB. Sedimentation equilibrium analysis indicated that, at neutral pH, TNB-bIg is monomeric while the intact protein is dimeric. In contrast, at pH 2.0, both the intact beta-lactoglobulin and TNB-bIg were monomeric. The unfolding transition of TNB-bIg induced by Gdn-HCl was cooperative in both pH regions, although the degree of cooperativity was less than that of the intact protein. The 1H-NMR spectrum for TNB-bIg at pH 3.0 was native-like, whereas the spectrum at pH 7.5 was similar to that of the unfolded proteins. These results suggest that modification of the buried thiol group destabilizes the rigid hydrophobic core and the dimer interface, producing a monomeric state that is native-like at pH 2.0 but is molten globule-like at pH 7.5. Upon reducing the mixed disulfide of TNB-bIg with dithiothreitol, the intact beta-lactoglobulin was regenerated. TNB-bIg will become a useful model to analyze the conformation and stability of the intermediate of protein folding.

Dataset Information

Protein self-association in solution: the bovine beta -lactoglobulin dimer and octamer.

Publications

Protein self-association in solution: the bovine beta -lactoglobulin dimer and octamer.

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