Designing conditions for in vitro formation of amyloid protofilaments and fibrils.
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.
Chiti
F
F
Oxford Centre for Molecular Sciences, New Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QT, United Kingdom.
Webster
P
P
Taddei
N
N
Clark
A
A
Stefani
M
M
Ramponi
G
G
Dobson
C M
CM
D017766:Acid Anhydride Hydrolases / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D000682:Amyloid / Q000096:biosynthesis*; D052160:Benzothiazoles; D001718:Birefringence; D002942:Circular Dichroism; D003224:Congo Red; D005456:Fluorescent Dyes; D006801:Humans; D008854:Microscopy, Electron; D018482:Muscle, Skeletal / Q000201:enzymology*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013844:Thiazoles; D000097024:Acylphosphatase
D017766:Acid Anhydride Hydrolases; D000682:Amyloid; D052160:Benzothiazoles; D003224:Congo Red; D005456:Fluorescent Dyes; D013844:Thiazoles; D000097024:Acylphosphatase; C009462:thioflavin T