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 pr otein aggregate (octamer/dimer) undergoes dissociation to form a dimer /monomer as the temperature is increased from 10 to 25/30 degrees C, a nd at temperatures above 25/30 degrees C, the protein undergoes a conf ormational change that leads to denaturation and aggregation/gelation. The dissociation and conformational change(s) occur in the fast excha nge regime on the NMR time scale. Calcium and Na+ do not induce format ion of different conformations in ''native'' beta-lactoglobulin B at 2 5 degrees C. The unfolded proteins adopt one or a few discrete conform ations in the presence of NaCl and CaCl2 that are different from the s tructure in H2O as the temperature is raised (to above 40 degrees C). 1H NMR kinetic experiments at 70 degrees C indicate that the folded fo rm unfolds within several minutes under all salt conditions and that s ubsequent aggregation and gel formation from the unfolded form involve s a slow step (several hours). Divalent cations apparently stabilize t he unfolded conformation by shifting the structural equilibrium from f olded to marginally unfolded and trapped by bound divalent cations. De uterium lock signal intensity changes, observed during the course of t hermal denaturation, suggest that protein aggregation/gelation occurs via different mechanisms in mono- and divalent ion solutions. Cd-111 N MR line widths indicate that the Cd2+ ions are not tightly bound to th e protein; Cd-111 NMR chemical shifts suggest that divalent cations bi nd to the protein predominantly at carboxylate oxygen sites (and proba bly to a limited extent at imidazole nitrogens).