Significance: Protein oligomers are a fundamental component of amyloidogenic disorders such as Alzheimer’s disease, being both critical intermediates in the formation of amyloid fibrils and the most toxic species on the protein aggregation pathway. Here, we derive general analytical expressions for the chemical kinetics of the oligomers generated during amyloid-formation reactions and explain how these may be used to categorize oligomers according to their basic physicochemical properties. By applying this framework to experimental kinetic data on the aggregation of a wide range of amyloidogenic proteins, we are able to determine features common to all amyloid oligomers. In particular, most are not capable of rapid growth by monomer addition, and most dissociate rather than mature into fibrils.

A general non-binary definition for on- and off-pathway intermediates is developed, enabling comparison of amyloid oligomers' contributions to fibril formation.

Oligomers of alpha-synuclein are toxic to cells and have been proposed to play a key role in the etiopathogenesis of Parkinson’s disease. As certain missense mutations in the gene encoding for alpha-synuclein induce early-onset forms of the disease, it has been suggested that these variants might have an inherent tendency to produce high concentrations of oligomers during aggregation, although a direct experimental evidence for this is still missing. We used single-molecule Förster Resonance Energy Transfer to visualize directly the protein self-assembly process by wild-type alpha-synuclein and A53T, A30P and E46K mutants and to compare the structural properties of the ensemble of oligomers generated. We found that the kinetics of oligomer formation correlates with the natural tendency of each variant to acquire beta-sheet structure. Moreover, A53T and A30P showed significant differences in the averaged FRET efficiency of one of the two types of oligomers formed compared to the wild-type oligomers, indicating possible structural variety among the ensemble of species generated. Importantly, we found similar concentrations of oligomers during the lag-phase of the aggregation of wild-type and mutated alpha-synuclein, suggesting that the properties of the ensemble of oligomers generated during self-assembly might be more relevant than their absolute concentration for triggering neurodegeneration.

The molecular mechanism of protein aggregation is of both fundamental and clinical importance as amyloid aggregates are linked to a number of neurodegenerative disorders. Such protein aggregates include macroscopic insoluble fibrils as well as small soluble oligomeric species. Time-dependent resolution of these species is prerequisite for a detailed quantitative understanding of protein aggregation; this remains challenging due to the lack of methods for detecting and characterizing transient and heterogeneous protein oligomers. Here we have used single molecule fluorescence techniques combined with mechanistic modelling to study the heparin-induced aggregation of the repeat region of tau, which forms the core region of neurofibrillary tangles found in Alzheimer’s disease. We distinguish several sub-populations of oligomers with different stability and follow their evolution during aggregation reactions as a function of temperature and concentration. Employment of techniques from chemical kinetics reveals that the two largest populations structurally distinct from fibrils; and are both kinetically and thermodynamically unstable. The first population is in rapid exchange with monomers and held together by electrostatic interactions; the second is kinetically more stable, dominates at later times and is probably off-pathway to fibril formation. These more stable oligomers may contribute to other oligomer induced effects in the cellular environment, for example by overloading protein quality control systems. We also show that the shortest growing filaments remain suspended in aqueous buffer and thus comprise a third, smaller population of transient oligomers with cross- structure. Overall our data show that a diverse population of oligomers of different structures and half-lives are formed during the aggregation reaction with the great majority of oligomers formed not going on to form fibrils.