Designing conditions for in vitro formation of amyloid protofilaments and fibrils

We have been able to convert a small alpha/beta protein, acylphosphatase, f rom its soluble and native form into insoluble amyloid fibrils of the type observed in a range of pathological conditions. This was achieved by allowi ng slow growth in a solution containing moderate concentrations of trifluor oethanol. When analyzed with electron microscopy, the protein aggregate pre sent in the sample after long incubation times consisted of extended, unbra nched filaments of 30-50 Angstrom in width that assemble subsequently into higher order structures. This fibrillar material possesses extensive beta-s heet structure as revealed by far-UV CD and IR spectroscopy. Furthermore, t he fibrils exhibit Congo red birefringence, increased fluorescence with thi oflavine T and cause a redshift of the Congo red absorption spectrum. All o f these characteristics are typical of amyloid fibrils. The results indicat e that formation of amyloid occurs when the native fold of a protein is des tabilized under conditions in which noncovalent interactions, and in partic ular hydrogen bonding, within the polypeptide chain remain favorable. We su ggest that amyloid formation is not restricted to a small number of protein sequences but is a property common to many, if not all, natural polypeptid e chains under appropriate conditions.