Structurally distinct amyloid protofibrils form on separate pathways of aggregation of a small protein

Abstract

Understanding the structural as well as mechanistic basis of the conformational polymorphism evident during amyloid protofibril and fibril formation by proteins is an important goal in the study of protein aggregation. In this report, we compare two separate routes to amyloid protofibril formation by the small protein barstar, one induced by the addition of trifluoroethanol (TFE) and the other by heat. The study reveals that the TFE-induced aggregation of barstar leads to protofibrils that differ from heat-induced protofibrils in their external dimensions and internal structures as well as in the mechanisms of their formation. Atomic force microscopy reveals that the TFE-induced protofibrils have about half the thickness of the heat-induced protofibrils. The thickness of the TFE-induced protofibrils (1.14±0.24) suggests that they form a β-sheet monolayer, while the thickness of the heat-induced protofibrils (2.56±0.32) suggests that they are built up from a pair (bilayer) of β-sheets. Fourier-transform infrared (FTIR) as well as circular dichroism (CD) spectroscopy shows that the heat-induced protofibrils are not pure β-sheet structures but that they also contain other structures (α-helix and/or random coil). In contrast, the TFE-induced protofibrils contain more β-sheet structures and less of other structures, if any. The FTIR and CD spectra also reveal that the two differently created protofibrils differ in the internal structures of their β-sheets. The TFE-induced protofibrils differ from the heat-induced protofibrils also in the kinetics of their formation. For the heat-induced reaction, the kinetics are monophasic without any lag phase, while the kinetics of the formation of TFE-induced protofibrils are sigmoidal with an initial lag phase. It appears that the TFE-induced and the heat-induced reactions involve distinct pathways for the formation of amyloid protofibrils. The existence of alternative pathways leading to amyloid protofibrils of distinct structures has important implications in understanding the kinetic origin of amyloid polymorphism.