Confinement-Induced Self-Assembly of Protein Nanofibrils Probed by Microfocus X-ray Scattering

We here explore confinement-induced assembly of whey protein nanofibrils (PNFs) into microscale fibers using microfocused synchrotron X-ray scattering. Solvent evaporation aligns the PNFs into anisotropic fibers, and the process is followed in situ by scattering experiments within a droplet of PNF dispersion. We find an optimal temperature at which the order parameter of the protein fiber is maximized, suggesting that the degree of order results from a balance between the time scales of the forced alignment and the rotational diffusion of the fibrils. Furthermore, the assembly process is shown to depend on the nanoscale morphology and flexibility of the PNFs. Stiff/straight PNFs with long persistence lengths (∼2 μm) align at the air-water interface, with anisotropy decreasing toward the center of the droplet as Marangoni flows increase entanglement toward the center. By contrast, flexible/curved PNFs with shorter persistence lengths (<100 nm) align more uniformly throughout the droplet, likely due to enhanced local entanglements. Straight PNFs pack tightly, forming smaller clusters with short intercluster distances, while curved PNFs form intricate, adaptable networks with larger characteristic distances and more varied structures.