Designing conditions for in vitro formation of amyloid protofilaments and fibrils

Abstract

We have been able to convert a small ␣͞␤ protein, acylphosphatase, from its soluble and native form into insoluble amyloid fibrils of the type observed in a range of pathological conditions. This was achieved by allowing slow growth in a solution containing moderate concentrations of trif luoroethanol. When analyzed with electron microscopy, the protein aggregate present in the sample after long incubation times consisted of extended, unbranched filaments of 30-50 Å in width that assemble subsequently into higher order structures. This fibrillar material possesses extensive ␤-sheet structure as revealed by far-UV CD and IR spectroscopy. Furthermore, the fibrils exhibit Congo red birefringence, increased f luorescence with thiof lavine T and cause a redshift of the Congo red absorption spectrum. All of these characteristics are typical of amyloid fibrils. The results indicate that formation of amyloid occurs when the native fold of a protein is destabilized under conditions in which noncovalent interactions, and in particular hydrogen bonding, within the polypeptide chain remain favorable. We suggest that 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 (1). These include Alzheimer's disease, type II diabetes, primary and secondary systemic amyloidosis, and the spongiform encephalopathies such as Creutzfeldt-Jakob disease. Although it is still debatable as to whether amyloid fibril deposition is itself the primary origin of the pathological condition with which it is associated, a variety of experimental observations suggests that there is a strong causal link between fibril formation and the onset of pathological symptoms (2, 3). Electron microscopy shows that the amyloid fibrils associated with the various diseases all appear straight and unbranched, and are 40-120 Å in diameter (4). X-ray fiber diffraction studies indicate a characteristic cross-␤ structure in which the polypeptide chains form ␤-strands oriented perpendicular to the long axis of the fibril, resulting in ␤-sheets propagating in the direction of the fibril (4). This overall structure is common to all amyloid fibrils despite the lack of sequence homologies among the amyloidogenic proteins and the absence of structural similarities in the folds that these proteins adopt under normal nonpathological conditions. This observation of a common structure for amyloid indicates that it 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 (1, 4). Recently, the SH3 domain of the p85␣ subunit of phosphatidylinositol 3-kinase, a protein that is not associated with any of the known amyloid diseases, was found readily to form amylo...

Solution structure and conformational heterogeneity of acylphosphatase from Bacillus subtilis

“…In addition, the NH 2 signals of Gln13 were absent and only one peak from the NH 2 of Asn36 was observed in the 2D 1 H-15 N HSQC spectrum. Overall, 91% of the backbone, and 90% of the side-chain 1 H, 15 N, and 13 C resonances were assigned for ligand-free BsAcP.…”

Spontaneous Fibril Formation by Polyalanines; Discontinuous Molecular Dynamics Simulations

“…Although little is known about the molecular basis for fibrillization, tantalizing clues emerge when the common features of the various amyloidoses are examined. Fibrils of amyloidogenic proteins formed in vitro exhibit strikingly similar morphologies despite a lack of similarity in their sequence, structure and function 4,[6][7][8][9] . They are invariably long, straight and unbranched, and consist of two or more smaller fibrils, called protofilaments (and sometimes protofibrils) which are themselves long ribbons of layered crossed β-sheets propagating along the fibril axis [10][11][12][13][14][15] .…”

Phosphorylation as Conformational Switch from the Native to Amyloid State: Trp-Cage as a Protein Aggregation Model

“…2A) and are similar to those reported for amyloid fibrils. [46][47][48][49] In case of D9E, the amplitude is relatively low which might be explained by the presence of significant amount of precipitated material with large aggregate particles. Some precipitates were also observed in the D9S aggregated sample, while aggregated 13S(P) was fully transparent showing a spectrum typical of amyloid fibrils.…”