The study of the aggregation of soluble proteins into highly ordered, insoluble amyloid fibrils is fundamental for the understanding of neurodegenerative disorders. Here, we present a method for the observation of single amyloid fibrils that allows the investigation of fibril growth, secondary nucleation or fibril breakup that is typically hidden in the average ensemble. Our approach of thermophoretic trapping and rotational diffusion measurements is demonstrated for single Aβ40, Aβ42 and pyroglutamyl-modified amyloid-β variant (pGlu3-Aβ3–40) amyloid fibrils.
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A dataset for demonstration can be downloaded at https://doi.org/10.5281/zenodo.1414296. The full dataset that supports the findings of this study is available from the corresponding author upon reasonable request.
The source code and the files for the software used in this study are contained in the Supplementary Software, and a maintained version can be downloaded at https://github.com/molecular-nanophotonics/thermophoretic-trap-for-protein-aggregation-studies.
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F.C. and D.H. acknowledge financial support by the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG) through the Collaborative Research Center TRR 102 ‘Polymers under multiple constraints: restricted and controlled molecular order and mobility’ (funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), project number 189853844–SFB TRR 102) and project CI 33/14-1. J.P. and M.M. acknowledge financial support by the BMBF (contract no. 03WKCL01G) and the DFG via the Cluster of Excellence ‘cfaed’ (contract no. EXC 1056/1). We thank A. Kramer for helping to revise the manuscript.
The authors declare no competing interests.
Peer review information: Allison Doerr was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Notes 1–12 and Supplementary Protocol
The repository contains a collection of Python scripts and Jupyter Notebooks for the tracking and data analysis of a single amyloid fibril in a thermophoretic trap.
Aβ40 fibril of length L = 1.5 μm confined inside the thermophoretic trap with an incident laser heating power of Pheat = 1 mW. A sample image from the movie is displayed in Fig. 1b of the main text. The dashed circle indicates the inner diameter (10 μm) of the trap. The exposure time and inverse framerate correspond to 30 ms.
Tracked position and orientation of the Aβ40 fibril shown in Supplementary Video 1 overplayed with the original video. The dashed circle indicates the inner diameter (10 μm) of the trap. The exposure time and inverse framerate correspond to 30 ms.
Fragmentation of a trapped Aβ40 fibril of length L = 1.1 μm corresponding to the image sequence Fig. 2f. The dashed circle indicates the inner diameter (10 μm) of the trap. The exposure time and inverse framerate correspond to 30 ms.
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Fränzl, M., Thalheim, T., Adler, J. et al. Thermophoretic trap for single amyloid fibril and protein aggregation studies. Nat Methods 16, 611–614 (2019). https://doi.org/10.1038/s41592-019-0451-6
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