Amyloid Fibril Formation in Solution and at Interfaces in Shearing Flows

Amyloid formation is a specific form of protein aggregation that refers to the self-assembly of proteins into fibrils following three distinct stages: protein unfolding, nuclei formation, and fibril growth. Previous observations have shown that shearing of protein solutions has a significant effect on fibrillization kinetics, but a clear understanding of the separate effects of hydrodynamic forces, convective transport of species, and interfaces on the fibrillization process is yet to be established. Studies here include fibrillization experiments in both quiescent and flowing conditions using insulin. Well-defined flow geometries (Poiseuille flow, Taylor-Couette with capillary confined end walls, and deep-channel surface viscometer) were employed in conjunction with optical fluorescence and bright field microscopy, atomic force microscopy, fluorescence and UV-Vis spectrophotometry, dynamic light scattering, and rheological measurements. Quiescent fibrillization constituted a baseline, but reproducibility of its kinetics was an issue. Preexisting high molecular weight amorphous protein aggregates were identified as the source of irreproducibility. Their role as "active centers", from which fibrils grow radially to form spherulites, reveals the importance that off-pathway amorphous aggregation has in determining quiescent fibrillization kinetics. The concentration of preexisting aggregates was diminished by pH cycling and by membrane filtration. It was found that filtration of insulin with large pore membranes generally slowed fibril formation relative to unfiltered solutions but, unexpectedly, filtration with small pore membranes showed no beneficial effects and, in some cases, accelerated insulin fibril formation. It was determined that filtration of aggregated species may have a detrimental effects due to fragmentation of aggregates during filtration, especially through small pore membranes. When flow is present, fibrillization is accelerated and, instead of the observed spherulites after quiescent fibrillization, mostly fibrils are formed. It was found that accelerated amyloid formation in sheared insulin solutions at low pH is caused mainly by hydrodynamic forces, rather than by interfacial effects or transport of species. The force field that the molecules experience significantly increases the amount of activated (unfolded) molecules and consequently the rate at which oligomers can form. As a second order effect, flow also accelerates the nucleation and growth stages, possibly by promoting molecular alignment. These findings reveal a useful analogy between amyloid formation and polymer crystallization. Novel encapsulated micro-bubbles, consisting of an air core stabilized by a shell comprised by amyloid fibrils, were formed as a result of shearing insulin solutions in the presence of abundant air/water interface. The amyloid formation process was accelerated at the air/water interface. However, species adsorbed at the air/water interface appeared to be "trapped" and unable to seed fibrillization in the bulk. The fibrillization process at quiescent and sheared air/water interfaces and its relation to amyloid formation in the bulk were studied using a deep-channel surface viscometer. Fibrillization was accelerated at the air/water interface under shearing and quiescent conditions. Surface shear viscosity was shown to be a useful rheological property to assess the progress of fibrillization at the interface. In quiescent experiments, amyloid formation in solution appeared to be independent from the earlier fibrillization that occurred at the air/water interface. Amyloid films formed at the air/water interfaces, with differences in morphology depending on whether the interface was sheared or not during the fibrillization process. In both cases, however, the films became rigid, with a consequent immobilization of the free surface. A novel portable flow apparatus was developed for in situ microscopy which provides uniform shear at the air/water interface and the bulk. The Taylor-Couette capillary apparatus uses surface tension to pin the contact line. This maintains the free surface at a fixed position in spite of changes in its gravitational orientation or fluid volume. The capabilities of the apparatus were demonstrated by studying bulk and interfacial amyloid formation in situ, via real-time fluorescence microscopy. By uniformly shearing both the air/water interface and the bulk, it was established that amyloid formation at the free surface occurs faster than in the sheared solution. A thin amyloid film forms at the interface which becomes immobile with time.