This study describes the in vitro formation of amyloid protofilaments and fibrils from the α/β protein acylphosphatase under specific conditions. The protein was converted from its soluble, native form into insoluble amyloid fibrils by allowing slow growth in a solution containing moderate concentrations of trifluoroethanol (TFE). Electron microscopy revealed extended, unbranched filaments of 30–50 Å in width, which assemble into higher order structures. The fibrillar material exhibited β-sheet structure as shown by far-UV CD and IR spectroscopy, Congo red birefringence, increased fluorescence with thioflavine T, and a redshift of the Congo red absorption spectrum, all typical of amyloid fibrils. The results suggest that amyloid formation occurs when the native fold of a protein is destabilized under conditions where noncovalent interactions, particularly hydrogen bonding, remain favorable. Amyloid formation is not restricted to a small number of protein sequences but is a property common to many, if not all, natural polypeptide chains under appropriate conditions. A range of human disorders is associated with the extracellular deposition of insoluble protein aggregates known as amyloid fibrils. These include Alzheimer's disease, type II diabetes, systemic amyloidosis, and spongiform encephalopathies. Although the role of amyloid fibril deposition in these diseases is still debated, experimental observations suggest a strong causal link between fibril formation and the onset of pathological symptoms. Amyloid fibrils are characterized by a common cross-β structure, where polypeptide chains form β-strands oriented perpendicular to the long axis of the fibril. This structure is common to all amyloid fibrils despite the lack of sequence homologies among the amyloidogenic proteins. The study shows that amyloid formation is stabilized by interactions associated with the common covalent structure of proteins, such as backbone hydrogen bonding or hydrophobic interactions, rather than through specific interactions of the different side chains. The study also demonstrates that the SH3 domain of the p85α subunit of phosphatidylinositol 3-kinase, a protein not associated with any known amyloid diseases, can form amyloid fibrils in vitro under acidic conditions. This prompted the exploration of whether other natural proteins might assemble into such fibrils under appropriate conditions. The study describes experiments to design such conditions using acylphosphatase as a model system. The results show that under appropriate conditions, this small protein forms a partially denatured state from which a protein aggregate containing amyloid protofilaments and fibrils develops slowly. The study also shows that the fibrillar material meets all criteria to be classified as amyloid, including Congo red birefringence, increased fluorescence with thioflavine T, and a redshift of the Congo red absorption spectrum. The findings suggest that amyloid formation is an intrinsicThis study describes the in vitro formation of amyloid protofilaments and fibrils from the α/β protein acylphosphatase under specific conditions. The protein was converted from its soluble, native form into insoluble amyloid fibrils by allowing slow growth in a solution containing moderate concentrations of trifluoroethanol (TFE). Electron microscopy revealed extended, unbranched filaments of 30–50 Å in width, which assemble into higher order structures. The fibrillar material exhibited β-sheet structure as shown by far-UV CD and IR spectroscopy, Congo red birefringence, increased fluorescence with thioflavine T, and a redshift of the Congo red absorption spectrum, all typical of amyloid fibrils. The results suggest that amyloid formation occurs when the native fold of a protein is destabilized under conditions where noncovalent interactions, particularly hydrogen bonding, remain favorable. Amyloid formation is not restricted to a small number of protein sequences but is a property common to many, if not all, natural polypeptide chains under appropriate conditions. A range of human disorders is associated with the extracellular deposition of insoluble protein aggregates known as amyloid fibrils. These include Alzheimer's disease, type II diabetes, systemic amyloidosis, and spongiform encephalopathies. Although the role of amyloid fibril deposition in these diseases is still debated, experimental observations suggest a strong causal link between fibril formation and the onset of pathological symptoms. Amyloid fibrils are characterized by a common cross-β structure, where polypeptide chains form β-strands oriented perpendicular to the long axis of the fibril. This structure is common to all amyloid fibrils despite the lack of sequence homologies among the amyloidogenic proteins. The study shows that amyloid formation is stabilized by interactions associated with the common covalent structure of proteins, such as backbone hydrogen bonding or hydrophobic interactions, rather than through specific interactions of the different side chains. The study also demonstrates that the SH3 domain of the p85α subunit of phosphatidylinositol 3-kinase, a protein not associated with any known amyloid diseases, can form amyloid fibrils in vitro under acidic conditions. This prompted the exploration of whether other natural proteins might assemble into such fibrils under appropriate conditions. The study describes experiments to design such conditions using acylphosphatase as a model system. The results show that under appropriate conditions, this small protein forms a partially denatured state from which a protein aggregate containing amyloid protofilaments and fibrils develops slowly. The study also shows that the fibrillar material meets all criteria to be classified as amyloid, including Congo red birefringence, increased fluorescence with thioflavine T, and a redshift of the Congo red absorption spectrum. The findings suggest that amyloid formation is an intrinsic