期刊
JOURNAL OF MOLECULAR BIOLOGY
卷 330, 期 4, 页码 785-797出版社
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/S0022-2836(03)00583-7
关键词
beta 2-microglobulin; thioflavin-T; atomic force microscopy; nucleation; assembly mechanism
资金
- Biotechnology and Biological Sciences Research Council [SF16972] Funding Source: Medline
- Biotechnology and Biological Sciences Research Council [SF16972] Funding Source: researchfish
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. (C) 2003 Elsevier Science Ltd. All rights reserved.
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