Fibrillation of β-lactoglobulin at pH 2.0: Impact of cysteine substitution and disulfide bond reduction

Fibrillar aggregation is a promising route to enhance protein functionality for food, while prefibrillar oligomers in vivo are suspected to be pathogenic. Here, the role of intramolecular disulfide bonds in fibrillation (80 °C, pH 2) of the major whey protein β-lactoglobulin (BLG) was studied. Different cysteine-modified variants were used for this investigation, removing one or both disulfide bonds in BLG. Denaturation occurred upon heating at pH 2.0 up to 1 h, but residual structure elements prevented aggregation of intact bovine BLG. Heating for longer caused acidic hydrolysis, which resulted in the release of peptides with enhanced aggregation tendency. Partial destabilisation by removal of C66–C160 (by recombinant substitution) or both disulfide bonds (by chemical cleavage) was found to affect this hydrolysis rate, but only cleavage of both disulfide bonds accelerate the aggregation kinetics. No major impact on the fibrillar morphology was observed. In contrast, recombinant removal of both disulfide bonds led to complete structural disruption and instantaneous aggregation of intact BLG at pH 2 prior to heating. These small aggregates (10–50 nm) led to the formation of worm-like aggregates within 1 h of heating, which slowed down the acidic hydrolysis and inhibited further aggregation into fibrils. We concluded that fibrillation can be accelerated by structural destabilisation through disulfide bond cleavage, while complete destabilisation can actually hinder it. These insights can be used for the production of functional protein aggregates, and possibly for the avoidance of pathogenic fibrillation of proteins.