title: Agitation and high ionic strength induce amyloidogenesis of a folded PDZ domain in native conditions.
abstract: Amyloid fibril formation is a distinctive hallmark of a number of degenerative diseases. In this process, protein monomers self-assemble to form insoluble structures that are generally referred to as amyloid fibrils. We have induced in vitro amyloid fibril formation of a PDZ domain by combining mechanical agitation and high ionic strength under conditions otherwise close to physiological (pH 7.0, 37 degrees C, no added denaturants). The resulting aggregates enhance the fluorescence of the thioflavin T dye via a sigmoidal kinetic profile. Both infrared spectroscopy and circular dichroism spectroscopy detect the formation of a largely intermolecular beta-sheet structure. Atomic force microscopy shows straight, rod-like fibrils that are similar in appearance and height to mature amyloid-like fibrils. Under these conditions, before aggregation, the protein domain adopts an essentially native-like structure and an even higher conformational stability (DeltaG(U-F)(H2O)). These results show a new method for converting initially folded proteins into amyloid-like aggregates. The methodological approach used here does not require denaturing conditions; rather, it couples agitation with a high ionic strength. Such an approach offers new opportunities to investigate protein aggregation under conditions in which a globular protein is initially folded, and to elucidate the physical forces that promote amyloid fibril formation.
authors.affiliation: Dipartimento di Scienze Biochimiche, Università di Firenze, Florence, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Sicorello
authors.forename: Silvia
authors.initials: S
authors.lastname: Torrassa
authors.forename: Gemma
authors.initials: G
authors.lastname: Soldi
authors.forename: Stefano
authors.initials: S
authors.lastname: Gianni
authors.forename: Carlo
authors.initials: C
authors.lastname: Travaglini-Allocatelli
authors.forename: Niccolò
authors.initials: N
authors.lastname: Taddei
authors.forename: Annalisa
authors.initials: A
authors.lastname: Relini
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Chiti
mesh_terms: D000682:Amyloid / Q000737:chemistry*; D000817:Anilino Naphthalenesulfonates; D000818:Animals; D052160:Benzothiazoles; D002942:Circular Dichroism; D005453:Fluorescence; D007700:Kinetics; D051379:Mice; D018625:Microscopy, Atomic Force; D008958:Models, Molecular; D009038:Motion; D009994:Osmolar Concentration; D054731:PDZ Domains; D017510:Protein Folding; D017433:Protein Structure, Secondary; D054595:Protein Tyrosine Phosphatase, Non-Receptor Type 13 / Q000737:chemistry* / Q000302:isolation & purification; D017550:Spectroscopy, Fourier Transform Infrared; D013844:Thiazoles
chemical_list: C515594:8-anilino-1-naphthalenesulfonic acid; D000682:Amyloid; D000817:Anilino Naphthalenesulfonates; D052160:Benzothiazoles; D013844:Thiazoles; C009462:thioflavin T; D054595:Protein Tyrosine Phosphatase, Non-Receptor Type 13