Abstract

Accumulation of insoluble protein fibrils with a characteristic cross-beta sheet structure is intimately associated with a multitude of human disorders ranging from Alzheimer’s and Parkinson’s disease to type-II diabetes. Understanding the molecular mechanisms regulating and promoting the formation of distinct species of amyloid aggregates in vitro and in vivo represents a critical step towards devising effective treatment strategies.

We have mapped out conditions for in vitro assembly of hen egg-white lysozyme into distinct amyloid species. Varying protein and salt concentrations, we observed three distinct growth regimes separated by sharp transition boundaries. At low salt/protein concentrations, “traditional” rigid fibrils formed. Upon crossing a salt- and protein-specific threshold, globular oligomers and their closely related curvilinear polymers emerged, which transitioned into ‘amorphous’ precipitation upon further increases in either solution parameter. These distinct assembly products displayed unique aggregation kinetics, morphologies and structural characteristics, as determined from in situ dynamic light scattering, fluorescence spectroscopy, atomic force microscopy and infrared spectroscopy. A colloidal model that accounts for protein charge repulsion and salt-mediated charge screening replicated both the sharp onset of oligomer formation and its dependence on solution conditions.

Using this phase diagram, we investigated the transition from oligomer-free to oligomeric fibril growth. Specifically we explored whether amyloid oligomers were precursors to, or off-pathway competitors of lysozyme fibril growth. We will present evidence that lysozyme oligomers, instead of promoting or competing with fibril formation, actively inhibit fibril nucleation in vitro. Such insight could prove important due to the critical role amyloid oligomers are believed to play in the etiology of amyloid diseases.