Denatured-State Conformation As Regulator of Amyloid Assembly Pathways?

Abstract

Deposits of insoluble protein fibrils with cross beta-sheet structure are the hallmark feature of numerous human disorders, including Alzheimer's disease and type II diabetes. Using correlated dynamic light scattering (DLS) and atomic force microscopy (AFM) we investigated amyloid formation with hen egg white lysozyme at acidic pH values. We found that there was a pronounced transition in the aggregation behavior at low vs near physiological salt concentrations. At low salt concentrations (< 100 mM), DLS indicated the near simultaneous nucleation of three distinct aggregate populations. For elevated salt concentrations, only a single aggregate peak nucleated. AFM imaging shortly after nucleation further indicated distinct aggregate morphologies and sizes. Low salt concentrations yielded polymeric aggregates of varying dimensions but consistent with the three aggregate peaks observed in DLS. Aggregation near physiological salt concentrations, in contrast, yielded oligomeric intermediates that nucleated into protofibril strands. It is commonly accepted that amyloid fibril growth by native proteins requires a partially denatured conformation. We wondered, however, whether these changes in aggregation behavior could be related to differences in the conformation of denatured lysozyme monomers. Using DLS to measure lysozyme's diffusivity, we found that lysozyme assumes a noticeably more extended conformation at low vs. high salt concentrations. These latter measurements were carefully corrected for the effects of protein interaction and variable solution viscosity on protein diffusivity. ANS fluorescence measurements revealed a similar trend towards increased solution exposure of lysozyme monomers at low salt concentrations compared to high salt concentrations. These observations suggest that amyloid fibril assembly pathways might depend on the conformation of the denatured state from which they grow. This has potentially significant implications for our understanding of amyloid fibril formation in general and how to control the emergence of toxic intermediates, in particular.