Evidence for a mechanism of amyloid formation involving molecular reorganisation within native-like precursor aggregates.
The aggregation of the alpha/beta protein acylphosphatase from Sulfolobus solfataricus has been studied under conditions in which the protein maintains a native-like, although destabilised, conformation and that therefore bear resemblance to a physiological medium. Static and dynamic light-scattering measurements indicate that under these conditions the protein aggregates rapidly, within two minutes. The initial aggregates are enzymatically active and have a secondary structure that is not yet characterized by the high content of cross-beta structure typical of amyloid, as inferred from Fourier transform infra-red and circular dichroism measurements. These species then convert slowly into enzymatically inactive aggregates that bind thioflavin T and Congo red, characteristic of amyloid structures, and contain extensive beta-sheet structure. Transmission electron microscopy reveals the presence in the latter aggregates of spherical species and thin, elongated protofibrils, both with diameters of 3-5 nm. Kinetic tests reveal that this process occurs without the need for dissolution and re-nucleation of the aggregates. Formation of thioflavin T-binding and beta-structured aggregates is substantially more rapid than unfolding of the native state, indicating that the initial aggregation process promotes formation of amyloid structures. Taken together, these findings suggest a mechanism of amyloid formation that may have physiological relevance and in which the amyloid structures result from reorganisation of the molecular interactions within the initially formed non-amyloid aggregates.
Plakoutsi
Georgia
G
Dipartimento di Scienze Biochimiche, Università di Firenze, Viale Morgagni 50, 50134 Firenze, Italy.
Bemporad
Francesco
F
Calamai
Martino
M
Taddei
Niccolò
N
Dobson
Christopher M
CM
Chiti
Fabrizio
F
D017766:Acid Anhydride Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D000682:Amyloid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D000817:Anilino Naphthalenesulfonates; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D002942:Circular Dichroism; D003224:Congo Red; D007700:Kinetics; D008854:Microscopy, Electron; D008958:Models, Molecular; D046912:Multiprotein Complexes; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D017550:Spectroscopy, Fourier Transform Infrared; D048229:Sulfolobus solfataricus / Q000201:enzymology* / Q000235:genetics; D014270:Trifluoroethanol; D020107:Troponin T; D000097024:Acylphosphatase
D017766:Acid Anhydride Hydrolases; D000682:Amyloid; D000817:Anilino Naphthalenesulfonates; D019843:Archaeal Proteins; D003224:Congo Red; D046912:Multiprotein Complexes; D011994:Recombinant Proteins; D014270:Trifluoroethanol; D020107:Troponin T; D000097024:Acylphosphatase; C027132:1-anilino-8-naphthalenesulfonate