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Title:

Hierarchical Assembly of beta2-Microglobulin Amyloid In Vitro Revealed by Atomic Force Microscopy

Date:

7/2003

Resource

Abstract:

The kinetics of spontaneous assembly of amyloid fibrils of wild-type beta2-microglobulin (beta2M) 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.

Content Emphasis

Peer Reviewed Journal Article

Target Audience

Technical Research

Citation:

Journal of Molecular Biology, Volume 330, Number 4, 18 July 2003, pp. 785-797

Publication:

Journal of Molecular Biology

Author:

Kad N.M.; Myers S.L.; Smith D.P.; Alastair Smith D.; Radford S.E.; Thomson N.H.

Volume:

330