Perturbation of the stability of amyloid fibrils through alteration of electrostatic interactions.

The self-assembly of proteins and peptides into polymeric amyloid fibrils is a process that has important implications ranging from the understanding of protein misfolding disorders to the discovery of novel nanobiomaterials. In this study, we probe the stability of fibrils prepared at pH 2.0 and composed of the protein insulin by manipulating electrostatic interactions within the fibril architecture. We demonstrate that strong electrostatic repulsion is sufficient to disrupt the hydrogen-bonded, cross-β network that links insulin molecules and ultimately results in fibril dissociation. The extent of this dissociation correlates well with predictions for colloidal models considering the net global charge of the polypeptide chain, although the kinetics of the process is regulated by the charge state of a single amino acid. We found the fibrils to be maximally stable under their formation conditions. Partial disruption of the cross-β network under conditions where the fibrils remain intact leads to a reduction in their stability. Together, these results support the contention that a major determinant of amyloid stability stems from the interactions in the structured core, and show how the control of electrostatic interactions can be used to characterize the factors that modulate fibril stability.

Relationship between prion propensity and the rates of individual molecular steps of fibril assembly.

Peptides and proteins possess an inherent propensity to self-assemble into generic fibrillar nanostructures known as amyloid fibrils, some of which are involved in medical conditions such as Alzheimer disease. In certain cases, such structures can self-propagate in living systems as prions and transmit characteristic traits to the host organism. The mechanisms that allow certain amyloid species but not others to function as prions are not fully understood. Much progress in understanding the prion phenomenon has been achieved through the study of prions in yeast as this system has proved to be experimentally highly tractable; but quantitative understanding of the biophysics and kinetics of the assembly process has remained challenging. Here, we explore the assembly of two closely related homologues of the Ure2p protein from Saccharomyces cerevisiae and Saccharomyces paradoxus, and by using a combination of kinetic theory with solution and biosensor assays, we are able to compare the rates of the individual microscopic steps of prion fibril assembly. We find that for these proteins the fragmentation rate is encoded in the structure of the seed fibrils, whereas the elongation rate is principally determined by the nature of the soluble precursor protein. Our results further reveal that fibrils that elongate faster but fracture less frequently can lose their ability to propagate as prions. These findings illuminate the connections between the in vitro aggregation of proteins and the in vivo proliferation of prions, and provide a framework for the quantitative understanding of the parameters governing the behavior of amyloid fibrils in normal and aberrant biological pathways.