Thermal Denaturation of β-Lactoglobulin. A 1H NMR Study

The conformational changes occurring in β-lactoglobulin when heated at pH 2 and 7.4 have been studied by 1 H NMR and deuterium exchange. At pH 2, much of the structure is preserved, and two-dimensional spectra can be obtained. Assigned NH resonances, belonging to different parts of the protein, were followed simultaneously as they disappeared from the spectrum upon heating at 45, 55, and 75 °C in 2 H 2 O. As judged by the extent of solvent deuterium exchange, denaturation occurred in stages. At 55 °C, strand E and the A−B loop unfolded. Strand A became partially flexible at 55 °C and lost the protective action of the α-helix at 75 °C, which became unfolded. At 75 °C, gelation occurred, with no observable opening of the β-barrel, although its internal face was partially exposed. The two blocks formed by the BCD and FGH β-sheets were very resistant to heat. The structural changes observed can be related to gelation, precipitation, and immunogenicity.