Nanopore analysis of amyloid fibrils formed by lysozyme aggregation.

Nikolay Martyushenko,Nicholas A W Bell,Robin D Lamboll,Ulrich F Keyser

doi:10.1039/c5an00530b

Nikolay Martyushenko, Nicholas A W Bell + Show 2 more

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https://doi.org/10.1039/c5an00530b

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Abstract

The measurement of single particle size distributions of amyloid fibrils is crucial for determining mechanisms of growth and toxicity. Nanopore sensing is an attractive solution for this problem since it gives information on aggregates' shapes with relatively high throughput for a single particle technology. In this paper we study the translocation of lysozyme fibrils through quartz glass nanopores. We demonstrate that, under appropriate salt and pH conditions, lysozyme fibrils translocate through bare quartz nanopores without causing significant clogging. This enables us to measure statistics on tens of thousands of translocations of lysozyme fibrils with the same nanopore and track their development over a time course of aggregation spanning 24 h. Analysis of our events shows that the statistics are consistent with a simple bulk conductivity model for the passage of rods with a fixed cross sectional area through a conical glass nanopore.

Highlights

Nanopores can obtain physical data about individual molecules and larger aggregates in solution and offer the advantage of not needing chemical labels
We observe an increase in fibril length over the time scale of incubation so that after 24 hours most fibrils are longer than the field of view which is a limitation of atomic force microscopy (AFM) analysis for amyloid fibrils
Having confirmed the formation of lysozyme fibrils by thioflavin T (ThT) fluorescence and AFM we investigated their translocation through quartz glass nanopores

Summary

Introduction

Nanopores can obtain physical data about individual molecules and larger aggregates in solution and offer the advantage of not needing chemical labels. Solid-state nanopores have potential as a platform for studying protein aggregation. Amyloids formed by aberrant protein aggregation are implicated in a wide range of diseases such as Alzheimer’s and Parkinson’s disease.[8] Amyloid fibrils are the archetypal amyloid state distinguished by their high aspect ratio together with cross β-sheet structure.[9] Accurate techniques for measuring the kinetics behind the formation of such amyloid fibrils are crucial for understanding the basis of their toxicity.[10] the study of amyloid fibrils is complicated due to the heterogeneity of sizes and non-linear rate laws for formation.[11] Single molecule techniques can

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