Tracing nucleation pathways in protein aggregation by using small angle scattering methods

Heat and alcohol-induced nucleation pathways of the whey protein β-lactoglobulin were investigated using small angle neutron and X-ray scattering for structural characterization. Protein solutions at various concentrations were either heated stepwise to 80 °C or mixed with ethanol to 50% (v/v). Heating induces dissociation of the β-lactoglobulin dimer in neutral pH aqueous medium, leading to nucleation at about 75 °C of tetrameric, cylindrical clusters, as indicated by three dimensional rigid body and bead modelling performed to fit scattering curves. In contrast to heating, ethanol addition induces the formation of fairly compact, internally disordered sphere-like clusters composed of rod-like submolecular structural units. At higher concentrations these clusters show typical colloidal behaviour, exhibiting long-range repulsive interactions, as also confirmed by dynamic light scattering measurements. The results contrast the effect of different unfolding scenarios on preferred nucleation pathways in subsequent protein assembly processes in various solution environments.