THERMAL-DENATURATION OF BETA-LACTOGLOBULIN - A H-1-NMR STUDY

The conformational changes occurring in beta-lactoglobulin when heated at pH 2 and 7.4 have been studied by H-1 NMR and deuterium exchange. At pH 2, much of the structure is preserved, and two-dimensional spect ra can be obtained. Assigned NH resonances, belonging to different par ts of the protein, were followed simultaneously as they disappeared fr om the spectrum upon heating at 45, 55, and 75 degrees C in (H2O)-H-2. As judged by the extent of solvent deuterium exchange, denaturation o ccurred in stages. At 55 degrees C, strand E and the A-B loop unfolded . Strand A became partially flexible at 55 degrees C and lost the prot ective action of the alpha-helix at 75 degrees C, which became unfolde d. At 75 degrees C, gelation occurred, with no observable opening of t he beta-barrel, although its internal face was partially exposed. The two blocks formed by the BCD and FGH beta-sheets were very resistant t o heat. The structural changes observed can be related to gelation, pr ecipitation, and immunogenicity.