Conformational prerequisites for alpha-lactalbumin fibrillation.

Bovine alpha-lactalbumin, a small acidic Ca(2+)-binding milk protein, formed amyloid fibrils at low pH, where it adopted the classical molten globule-like conformation. Fibrillation was accompanied by a dramatic increase in the beta-structure content and a characteristic increase in the thioflavin T fluorescence intensity. S-(Carboxymethyl)-alpha-lactalbumin, a disordered form of the protein with three out of four disulfide bridges reduced, was even more susceptible to fibrillation. Other partially folded conformations induced in the intact protein at neutral pH, either by the removal of Ca(2+) or by the binding of Zn(2+) to the Ca(2+)-protein complex, did not fibrillate, although Zn(2+)-loaded alpha-lactalbumin precipitated out of solution as amorphous aggregates. Our data suggest that the transformation of a protein into an essentially unfolded (thus, highly flexible) conformation is required for successful fibril formation, whereas more rigid (but still flexible) species may form amorphous aggregates.