Effect of Hydrophobic Residues on Interfacial Fibrillization Kinetics

Abstract

The presence of amyloid fibril plaques in the brain is strongly associated with several neurological disorders. Prediction of fibril formation is a daunting challenge because of the large number of routes to formation, the sensitivity of protein structures to various stresses, differences in fibrillization propensities between proteins, and the vast number of possible amyloid structures. Bovine insulin differs from human by replacement of just three amino acids, yet this difference is sufficient to result in vastly altered fibrillization kinetics. One possibility for this large deviation may be the hydrophobicity of the three substituted residues. Quantification of these differences can provide clues into how and why fluid flows and interfaces influence protein stability and induce aggregation. The exact roles of shear flow, mixing, and interfacial phenomena on fibrillization are difficult to distinguish because these effects have been coupled in previous experiments. In this work, precision flow devices are used to decouple such effects. Mixing and shear flow have been separated using a combination of a rotating Couette flow and solid body rotation. Interfacial effects are determined by kinetic differences between flows with and without the air-water interface and by using a knife-edge viscometer to investigate the effects of interfacial shear. Together, these flow geometries and boundary conditions can be used to distinguish the individual effects of different fluid phenomena. Comparing flow and interfacial effects on fibrillization time between human and bovine insulin facilitates determination of the roles amino residues play in destabilization and aggregation. These findings have important implications for protein stability at interfaces and within biophysically relevant flow fields.