title: Hierarchical assembly of beta2-microglobulin amyloid in vitro revealed by atomic force microscopy.
abstract: The kinetics of spontaneous assembly of amyloid fibrils of wild-type beta(2)-microglobulin (beta(2)M) in vitro, under acid conditions (pH 2.5) and low ionic strength, has been followed using thioflavin-T (ThT) binding. In parallel experiments, the morphology of the different fibrillar species present at different time-points during the growth process were characterised using tapping-mode atomic force microscopy (TM-AFM) in air and negative stain electron microscopy (EM). The thioflavin-T assay shows a characteristic lag phase during which the nucleation of fibrils occurs before a rapid growth in fibril density. The volume of fibrils deposited on mica measured from TM-AFM images at each time-point correlates well with the fluorescence data. TM-AFM and negative-stain EM revealed the presence of various kinds of protein aggregates in the lag phase that disappear concomitantly with a rise in the density of amyloid fibrils, suggesting that these aggregates precede fibril growth and may act as nucleation sites. Three distinct morphologies of mature amyloid fibrils were observed within a single growth experiment, as observed previously for the wild-type protein and the variant N17D. Additional supercoiled morphologies of the lower-order fibrils were observed. Comparative height analysis from the TM-AFM data allows each of the mature fibril types and single protofilaments to be identified unambiguously, and reveals that the assembly occurs via a hierarchy of morphological states.
authors.affiliation: School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
authors.forename: Neil M
authors.initials: NM
authors.lastname: Kad
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Myers
authors.forename: David P
authors.initials: DP
authors.lastname: Smith
authors.forename: D Alastair
authors.initials: DA
authors.lastname: Smith
authors.forename: Sheena E
authors.initials: SE
authors.lastname: Radford
authors.forename: Neil H
authors.initials: NH
authors.lastname: Thomson
mesh_terms: D000682:Amyloid / Q000737:chemistry*; D002455:Cell Division; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D018625:Microscopy, Atomic Force; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry; D013997:Time Factors; D001613:beta 2-Microglobulin / Q000737:chemistry*
chemical_list: D000682:Amyloid; D011994:Recombinant Proteins; D001613:beta 2-Microglobulin