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A diverse range of proteins can assemble into amyloid fibrils, a process that generally results in a loss of function and an increase in toxicity. The occurrence and rate of conversion is strongly dependent on several factors including molecular structure and exposure to hydrodynamic forces. To investigate the origins of shear-induced enhancement in the rate of fibrillization, a stable rotating Couette flow was used to evaluate the kinetics of amyloid formation under uniform shear for two similar insulin species (human and bovine) that demonstrate unique fibrillization kinetics. The presence of shear-induced nuclei predicted by previous studies is supported by observations of a lag between the consumption of soluble insulin and the precipitation of amyloid aggregates. The apparent fibrillization rate generally increases with shear. However, a two-parameter kinetic model revealed that the nucleation rate has a maximum value at intermediate shear rates. The fibril elongation rate increases monotonically with shear and is similar for both insulin variants, suggesting that increased elongation rates are related to mixing. Differences between human and bovine insulin kinetics under shear are attributable to the nucleation step.

Amyloidogenesis of proteins is of wide interest because amyloid structures are associated with many diseases, including Alzheimer's and type II diabetes. Dozens of different proteins of various sizes are known to form amyloid fibrils. While there are numerous studies on the fibrillization of insulin induced by various perturbations, shearing at fluid interfaces has not received as much attention. Here, we present a study of human insulin fibrillization at room temperature using a deep-channel surface viscometer. The hydrodynamics of the bulk flow equilibrates in just over a minute, but the proteins at the air-water interface exhibit a very slow development during which the surface (excess) shear viscosity deduced from a Newtonian surface model increases slightly over a period of a day and a half. Then, there is a very rapid increase in the surface shear viscosity to effectively unbounded levels as the interface becomes immobilized. Atomic force microscopy shows that fibrils appear at the interface after it becomes immobilized. Fibrillization in the bulk does not occur until much later. This has been verified by concurrent atomic force microscopy and circular dichroism spectroscopy of samples from the bulk. The immobilized interface has zero in-plane shear rate, however due to the bulk flow, there is an increase in the strength of the normal component of the shear rate at the interface, implicating this component of shear in the fibrillization process ultimately resulting in a thick weave of fibrils on the interface. Real-time detection of fibrillization via interfacial rheology may find utility in other studies of proteins at sheared interfaces.

Agitation of protein is known to induce deleterious effects on protein stability and structure, with extreme agitation sometimes resulting in complete aggregation into amyloid fibrils. Many mechanisms have been proposed to explain how protein becomes unstable when subjected to flow, including alignment of protein species, shear-induced unfolding, simple mixing, or fragmentation of existing fibrils to create new seeds. Here a shearing flow was imposed on a solution of monomeric human insulin via a rotating Couette device with a small hydrophobic fluid interface. The results indicate that even very low levels of shear are capable of accelerating amyloid fibril formation. Simulations of the flow suggest that the shear enhances fibrillization kinetics when flow inertia is non-negligible and the resulting meridional circulation allows for advection of bulk protein to the hydrophobic interface.