Designing conditions for in vitro formation of amyloid protofilaments and fibrils

Abstract

We have been able to convert a small alpha/beta 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 trifluoroethanol. When analyzed with electron microscopy, the protein aggregate present in the sample after long incubation times consisted of extended, unbranched filaments of 30-50 A in width that assemble subsequently into higher order structures. This fibrillar material possesses extensive beta-sheet structure as revealed by far-UV CD and IR spectroscopy. Furthermore, the fibrils exhibit Congo red birefringence, increased fluorescence with thioflavine T and cause a red-shift 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.

Figure 1

(A) Far-UV CD spectra of muscle acylphosphatase acquired during the aggregation process. The first and last spectra reported in the figure were acquired after 3 min and 10 hr after the initiation of the reaction, respectively. The spectra show a slow two-state transition between two conformations containing significant amounts of α-helical and β-sheet structure, respectively. After 10 hr of incubation, the spectra do not change their appearance, but undergo a progressive reduction of signal and a shift of the negative peak toward the higher wavelengths, as a result of the accumulation of protein aggregates. (B) Amide I region of the IR spectrum of muscle acylphosphatase denatured in TFE. The spectrum was recorded after a period of 2 hr from the start of the reaction. The two peaks at 1,613 and 1,685 cm⁻¹ are indicative of intermolecular β structure (19).

Figure 2

Electron micrographs showing the morphological development of acylphosphatase aggregates. (A) Aggregate of granular aspect formed 72 min after initiation of the reaction. (B) Short fibrils formed after 32 hr. (C) Protofilaments formed after 14 days. (D) Clusters of protofilaments formed after 45 days. In C, the two micrographs show fibrils from two different preparations. (Bar = 0.05 μm.)

Figure 3

Optical microscope image obtained under cross-polarized light and containing acylphosphatase aggregates stained with Congo red. The protein aggregate was obtained after 14 days of incubation in 25% (vol/vol) TFE. The photograph shows the blots of green birefringence coming from regions rich in amyloid fibrils.