Structural Features and Reversible Association of Different Quaternary Structures of β-Lactoglobulin

Structural and functional features were studied on the native dimeric form of β-lactoglobulin at neutral pH and on the monomeric forms obtained either by raising the pH to 9.0 or by blocking the thiol group of Cys121 with iodoacetamide under bland dissociating conditions. The thiol blocked monomer did not reassociate to native-like dimers, it retained retinol-binding ability, and it was found to display many of the structural features of the monomer obtained at pH 9, but differed in several structural features from the native dimer. The supramolecular associative properties of the proteins were studied by measuring concentration dependence of the accessibility of the backbone exchangeable amide protons in 1H NMR H/D exchange experiments, of their ligand-binding properties, and of their intrinsic fluorescence features. Evidence of reversible association was found for all the proteins with a very similar concentration dependence, indicating that the weak forces involved in this association were different from those stabilizing the native dimer.