Phase Boundaries for Fibril and Metastable Oligomer Formation of Lysozyme

Abstract

Deposition of protein fibers with a characteristic cross-beta sheet structure is the molecular marker for many human disorders including Alzheimer's disease, type II diabetes and rheumatoid arthritis. Given the large number of proteins and peptides beyond those associated with diseases that have been shown to form amyloid fibrils in vitro, it has been suggested that amyloid fibril formation represents a generic protein phase transition. Mapping out the corresponding phase boundaries is complicated by the presence of least two distinct fibril assembly pathways. One pathway is characterized by the nucleation of long, rigid fibrils common to the late stages of amyloid diseases. A second pathway involves the formation of globular oligomeric species and curvilinear protofibril. The relation of this latter pathway to the mature fibrils is not entirely clear. Intriguingly, it is these latter oligomers and protofibrils that have been implicated as the molecular species mediating the cellular toxicity associated with amyloid diseases.For the amyloidogenic protein lysozyme, we have systematically mapped out the combination of protein and salt concentrations resulting in the formation of either long rigid or oligomeric amyloid aggregates for fixed temperature and pH. Using dynamic light scattering, thioflavin fluorescence spectroscopy, atomic force microscopy and infrared spectroscopy, we detected three distinct types of aggregates. Growth of long straight fibrils prevailed at either low salt or protein concentrations. At intermediate salt and protein concentration oligomer formation with subsequent protofibril nucleation prevailed. Oligomers and protofibrils represent metastable phases that are kinetically favored, while long straight fibrils are the thermodynamically stable state. Eventually, fibril formation gives way to amorphous precipitation. This phase behavior shows intriguing similarities with the phase diagram for protein crystallization where a metastable liquid-liquid phase is located within the stable coexistence region for protein crystals.