title: Amyloid formation from HypF-N under conditions in which the protein is initially in its native state.
abstract: Aggregation of the N-terminal domain of the Escherichia coli HypF (HypF-N) was investigated in mild denaturing conditions, generated by addition of 6-12% (v/v) trifluoroethanol (TFE). Atomic force microscopy indicates that under these conditions HypF-N converts into the same type of protofibrillar aggregates previously shown to be highly toxic to cultured cells. These convert subsequently, after some weeks, into well-defined fibrillar structures. The rate of protofibril formation, monitored by thioflavin T (ThT) fluorescence, depends strongly on the concentration of TFE. Prior to aggregation the protein has far-UV circular dichroism (CD) and intrinsic fluorescence spectra identical with those observed for the native protein in the absence of co-solvent; the quenching of the intrinsic tryptophan fluorescence by acrylamide and the ANS binding properties are also identical in the two cases. These findings indicate that HypF-N is capable of forming amyloid protofibrils and fibrils under conditions in which the protein is initially in a predominantly native-like conformation. The rate constants for folding and unfolding of HypF-N, determined in 10% TFE using the stopped-flow technique, indicate that a partially folded state is in rapid equilibrium with the native state and populated to ca 1%. A kinetic analysis reveals that aggregation results from molecules accessing such a partially folded state. The approach described here shows that it is possible to probe the mechanism of aggregation of a specific protein under conditions in which the protein is initially native and hence relevant to a physiological environment. In addition, the results indicate that toxic protofibrils can be formed from globular proteins under conditions that are only marginally destabilising and in which the large majority of molecules have the native fold. This conclusion emphasises the importance for cells to constantly combat the propensity for even the most stable of these proteins to aggregate.
authors.affiliation: Dipartimento di Scienze Biochimiche, Università di Firenze, Viale Morgagni 50, 50134 Firenze, Italy.
authors.forename: Giordana
authors.initials: G
authors.lastname: Marcon
authors.forename: Georgia
authors.initials: G
authors.lastname: Plakoutsi
authors.forename: Claudio
authors.initials: C
authors.lastname: Canale
authors.forename: Annalisa
authors.initials: A
authors.lastname: Relini
authors.forename: Niccolò
authors.initials: N
authors.lastname: Taddei
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Dobson
authors.forename: Giampietro
authors.initials: G
authors.lastname: Ramponi
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Chiti
mesh_terms: D000682:Amyloid / Q000096:biosynthesis*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D006801:Humans; D018625:Microscopy, Atomic Force; D008958:Models, Molecular; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding
chemical_list: D000682:Amyloid; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C108878:HypF protein, Bacteria