Role of calcium as trigger in thermal beta-lactoglobulin aggregation.
Divalent calcium ions have been suggested to be involved in intermolecular protein-Ca2+-protein cross-linking, intramolecular electrostatic shielding, or ion-induced protein conformational changes as a trigger for protein aggregation at elevated temperatures. To address the first two phenomena in the case of beta-lactoglobulin, a combination of chemical protein modification, calcium-binding, and aggregation studies was used, while the structural integrity of the modified proteins was maintained. Although increasing the number of carboxylates on the protein by succinylation results in improved calcium-binding, calcium appears to be less effective in inducing protein aggregation. In fact, the larger the number of carboxylates, the higher the concentration of calcium that is required to trigger the aggregation. Lowering the number of negative charges on the protein surface via methylation of carboxylates reduces calcium-binding properties, but calcium-induced aggregation at low concentration is improved. Monovalent sodium ions cannot take over the specific role of calcium. The relation between net surface charge and number of calcium ions bound required to trigger the aggregation suggests that calcium needs to bind site specific to carboxylates with a threshold affinity. Subsequent site-specific screening of surface charges results in protein aggregation, driven by the partial unfolding of the protein at elevated temperatures, which is then facilitated by the absence of electrostatic repulsion.
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology*; D002413:Cations, Divalent / Q000494:pharmacology; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007202:Indicators and Reagents; D007700:Kinetics; D007782:Lactoglobulins / Q000737:chemistry* / Q000187:drug effects / Q000302:isolation & purification; D008892:Milk; D013499:Surface Properties
D002413:Cations, Divalent; D007202:Indicators and Reagents; D007782:Lactoglobulins; D002118:Calcium