Structural features of transiently modified beta-lactoglobulin relevant to the stable binding of large hydrophobic molecules

Binding sites for hydrophobic molecules on bovine (beta)-lactoglobulin, and their susceptibility to temperature, were studied by using various spectroscopic probes. Binding of probes carrying a single fluorophore moiety, a single nitroxide moiety, or both moieties on the same molecule, was followed by EPR and fluorescence. The presence of a fatty acid side chain in the dual probes was found to be required for binding to (beta)-lactoglobulin. Binding occurred only after the protein was heated at temperatures below the threshold for its irreversible denaturation. Binding became extremely tight and stable upon cooling of the protein-probe mixture. Comparison among the various probes suggests that multiple binding sites for hydrophobes are present in the native protein, and in the partially-and reversibly-modified form of (beta)-lactoglobulin present in solution at neutral pH and subdenaturing temperatures. Thus, the specificity of hydrophobes binding to (beta)-lactoglobulin may be modulated by simple physical treatment of the protein.