Amyloid-like aggregation of recombinant β-lactoglobulin at pH 3.5 and 7.0: Is disulfide bond removal the key to fibrillation?

Functional nanofibrils from globular proteins are usually formed by heating for several hours at pH 2.0, which induces acidic hydrolysis and consecutive self-association. The functional properties of these micro-metre-long anisotropic structures are promising for biodegradable biomaterials and food applications, but their stability at pH > 2.0 is low. The results presented here show that modified β-lactoglobulin can also form nanofibrils by heating at neutral pH without prior acidic hydrolysis; the key is removing covalent disulfide bonds via precision fermentation. The aggregation behaviour of various recombinant β-lactoglobulin variants was systemically studied at pH 3.5 and 7.0. The suppression of intra- and intermolecular disulfide bonds by eliminating one to three out of the five cysteines makes the non-covalent interactions more prevalent and allow for structural rearrangement. This stimulated the linear growth of worm-like aggregates. Full elimination of all five cysteines led to the transformation of worm-like aggregates into actual fibril structures (several hundreds of nanometres long) at pH 7.0. This understanding of the role of cysteine in protein-protein interactions will help to identify proteins and protein modifications to form functional aggregates at neutral pH.