Graduation Year

2008

Document Type

Thesis

Degree

M.S.

Degree Granting Department

Physics

Major Professor

Martin Muschol, Ph.D.

Keywords

Dynamic light scattering, Atomic force microcopy, Lysozyme, Protofibril nucleation, Oligomer

Abstract

The mechanisms linking deposits of insoluble fibrils of amyloid proteins to the debilitating neuronal cell death characteristic of neurodegenerative diseases remain enigmatic. Recent findings suggest that transiently formed intermediate aggregates, and not the prominent neuronal plaques, represent the principal toxic agent. Evaluating the neurotoxicity of intermediate aggregates, however, requires unambiguous characterization of all aggregate structures present, their relative distributions, and how they evolve in time. Hen-egg white lysozyme represents an attractive model for studying intermediate aggregate formation since it is an extensively characterized globular protein, and its human variants can lead to systemic amyloidosis. Combining in-situ dynamic light scattering (DLS) with atomic force microscopy (AFM), we have characterized the morphologies and growth kinetics of intermediate aggregates formed during lysozyme fibrillogenesis. Upon incubation at elevated temperatures, small uniform oligomers form with their numbers increasing for several hours. After a variable lag period protofibrils spontaneously nucleate. The heights and widths of protofibrils closely match those of oligomers. This match in physical dimensions, combined with the delayed onset of protofibril nucleation vs. the continuous formation of oligomers, suggest that protofibrils both nucleate and grow from oligomers. Protofibril morphologies and structures, visualized with AFM, are quite distinct from subsequently emerging mature fibrils. Overall, the evolution of aggregate morphologies during lysozyme fibrillogenesis follows a clear hierarchical pathway: amyloid monomers initially coalesce into oligomers of uniform size. Their steadily increasing numbers eventually induce nucleation and growth of protofibrils. Protofibrils, in turn, nucleate and grow via oligomer addition until they start to self-assemble into micron-sized double-stranded fibrils.

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