title: Direct characterization of amyloidogenic oligomers by single-molecule fluorescence.
abstract: A key issue in understanding the pathogenic conditions associated with the aberrant aggregation of misfolded proteins is the identification and characterization of species formed during the aggregation process. Probing the nature of such species has, however, proved to be extremely challenging to conventional techniques because of their transient and heterogeneous character. We describe here the application of a two-color single-molecule fluorescence technique to examine the assembly of oligomeric species formed during the aggregation of the SH3 domain of PI3 kinase. The single-molecule experiments show that the species formed at the stage of the reaction where aggregates have previously been found to be maximally cytotoxic are a heterogeneous ensemble of oligomers with a median size of 38 +/- 10 molecules. This number is remarkably similar to estimates from bulk measurements of the critical size of species observed to seed ordered fibril formation and of the most infective form of prion particles. Moreover, although the size distribution of the SH3 oligomers remains virtually constant as the time of aggregation increases, their stability increases substantially. These findings together provide direct evidence for a general mechanism of amyloid aggregation in which the stable cross-beta structure emerges via internal reorganization of disordered oligomers formed during the lag phase of the self-assembly reaction.
authors.affiliation: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom.
authors.forename: Angel
authors.initials: A
authors.lastname: Orte
authors.forename: Neil R
authors.initials: NR
authors.lastname: Birkett
authors.forename: Richard W
authors.initials: RW
authors.lastname: Clarke
authors.forename: Glyn L
authors.initials: GL
authors.lastname: Devlin
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Dobson
authors.forename: David
authors.initials: D
authors.lastname: Klenerman
mesh_terms: D000682:Amyloid / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D005453:Fluorescence; D007700:Kinetics; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry / Q000378:metabolism; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D018909:src Homology Domains
chemical_list: D000682:Amyloid; D019869:Phosphatidylinositol 3-Kinases
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