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Hierarchical assembly of beta2-microglobulin amyloid in vitro revealed by atomic force microscopy
Neil M Kad1, Sarah L Myers, David P Smith
1School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
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.
Insights
This study tracks amyloid fibril formation from beta(2)-microglobulin using thioflavin-T and microscopy. It reveals protein aggregates precede fibril growth and identifies a hierarchy of fibril structures during assembly.
Area of Science:
- Biochemistry
- Structural Biology
- Biophysics
Background:
- Amyloid fibrils are associated with various diseases.
- Understanding the assembly kinetics and morphology of amyloid fibrils is crucial for disease mechanism studies.
- Beta(2)-microglobulin (beta(2)M) is a protein prone to forming amyloid fibrils.
Purpose of the Study:
- To investigate the kinetics and morphology of spontaneous amyloid fibril assembly of wild-type beta(2)M in vitro.
- To characterize the intermediate species and fibril structures formed during the assembly process.
- To elucidate the hierarchical nature of amyloid fibril formation.
Main Methods:
- Thioflavin-T (ThT) binding assay to monitor fibril formation kinetics.
- Tapping-mode atomic force microscopy (TM-AFM) to analyze fibril morphology and volume.
- Negative stain electron microscopy (EM) for high-resolution structural characterization.
Main Results:
- A characteristic lag phase was observed, indicating a nucleation-dependent process.
- Protein aggregates were identified in the lag phase, suggesting they act as nucleation sites.
- Multiple distinct fibril morphologies, including supercoiled structures, were observed, indicating a hierarchy of assembly states.
Conclusions:
- The study provides insights into the early stages of amyloid fibril formation, highlighting the role of intermediate aggregates.
- TM-AFM and EM reveal a complex hierarchy of morphological states during beta(2)M amyloid assembly.
- These findings contribute to understanding the structural diversity and assembly pathways of amyloid fibrils.