NMR-STUDIES OF THERMAL-DENATURATION AND CATION-MEDIATED AGGREGATION OF BETA-LACTOGLOBULIN

Thermal denaturation, aggregation, and gelation of beta-lactoglobulin in solutions containing either no additional salt, 100 mM NaCl, or 20 mM CaCl2 were investigated using H-1 and Cd-111 NMR spectroscopies. H-1 NMR temperature dependence experiments suggest that an oligomeric protein aggregate (octamer/dimer) undergoes dissociation to form a dimer/monomer as the temperature is increased from 10 to 25/30 degrees C, and at temperatures above 25/30 degrees C, the protein undergoes a conformational change that leads to denaturation and aggregation/gelation. The dissociation and conformational change(s) occur in the fast exchange regime on the NMR time scale. Calcium and Na+ do not induce formation of different conformations in ''native'' beta-lactoglobulin B at 25 degrees C. The unfolded proteins adopt one or a few discrete conformations in the presence of NaCl and CaCl2 that are different from the structure in H2O as the temperature is raised (to above 40 degrees C). 1H NMR kinetic experiments at 70 degrees C indicate that the folded form unfolds within several minutes under all salt conditions and that subsequent aggregation and gel formation from the unfolded form involves a slow step (several hours). Divalent cations apparently stabilize the unfolded conformation by shifting the structural equilibrium from folded to marginally unfolded and trapped by bound divalent cations. Deuterium lock signal intensity changes, observed during the course of thermal denaturation, suggest that protein aggregation/gelation occurs via different mechanisms in mono- and divalent ion solutions. Cd-111 NMR line widths indicate that the Cd2+ ions are not tightly bound to the protein; Cd-111 NMR chemical shifts suggest that divalent cations bind to the protein predominantly at carboxylate oxygen sites (and probably to a limited extent at imidazole nitrogens).