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Hierarchical Assembly of beta2-Microglobulin Amyloid In Vitro Revealed by Atomic Force Microscopy
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.
Peer Reviewed Journal Article
Journal of Molecular Biology, Volume 330, Number 4, 18 July 2003, pp. 785-797
Journal of Molecular Biology
Kad N.M.; Myers S.L.; Smith D.P.; Alastair Smith D.; Radford S.E.; Thomson N.H.
Last updated on April 27, 2007
This work is supported in part by the Nanoscale Science and Engineering Initiative of the National Science Foundation
under NSF Award Number EEC-0118007.
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