Snapshots of fibrillation and aggregation kinetics in multistranded amyloid β-lactoglobulin fibrils

We have investigated the structural time-evolution of multistranded beta-lactoglobulin protein fibrils at pH 2 and 90 degrees C by combining small angle neutron scattering (SANS), dynamic (DLS) and depolarized light scattering (DDLS) as well as atomic force microscopy (AFM). Light scattering techniques, combined with SANS clearly demonstrate the different stages of conversion of beta-lactoglobulin monomers (2 wt %) into semiflexible protein fibrils upon heating at 90 degrees C. In addition, atomic force microscopy allows the resolution of some details of the fibrils at the molecular length scale which bulk scattering techniques cannot capture. Thus, we were able to resolve and identify different individual stages of the fibrillation process, including the formation of protofilaments, their alignment and aggregation into mature multistranded fibrils, and the development of a periodic pitch along their contour length. The picture emerging from combination of the scattering and single molecule techniques is consistent with three critical steps: (i) individual protofilaments align upon approaching due to liquid crystalline interactions; (ii) short range attractions among filaments-presumably of Lennard-Jones or hydrophobicity type-lead to irreversible aggregation of nearly perfectly aligned protofilaments into multistranded ribbon-like fibrils; (iii) intramolecular electrostatic repulsion of fibrils leads to twisting of the ribbon along the axis leading to the development of a periodic pitch along the fibrils contour length. The individual stages of the fibrillation and aggregation process are discussed in detail, in terms of the colloidal physics involved.