Thermodynamic characterization of amyloid polymorphism by microfluidic transient incomplete separation

Azad Farzadfard, Antonin Kunka, Thomas Oliver Mason, Jacob Aunstrup Larsen, Rasmus Krogh Norrild, Elisa Torrescasana Dominguez, Soumik Ray, Alexander K. Buell*

*Corresponding author for this work

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Abstract

Amyloid fibrils of proteins such as α-synuclein are a hallmark of neurodegenerative diseases and much research has focused on their kinetics and mechanisms of formation. The question as to the thermodynamic stability of such structures has received much less attention. Here, we newly utilize the principle of transient incomplete separation of species in laminar flow in combination with chemical depolymerization for the quantification of amyloid fibril stability. The relative concentrations of fibrils and monomer at equilibrium are determined through an in situ separation of these species based on their different diffusivity inside a microfluidic capillary. The method is highly sample economical, using much less than a microliter of sample per data point and its only requirement is the presence of aromatic residues (W, Y) because of its label-free nature, which makes it widely applicable. Using this method, we investigate the differences in thermodynamic stability between different fibril polymorphs of α-synuclein and quantify these differences for the first time. Importantly, we show that fibril formation can be under kinetic or thermodynamic control and that a change in solution conditions can both stabilise and destabilise amyloid fibrils. Taken together, our results establish the thermodynamic stability as a well-defined and key parameter that can contribute towards a better understanding of the physiological roles of amyloid fibril polymorphism.
Original languageEnglish
JournalChemical Science
Volume15
Issue number7
Pages (from-to)2528-2544
ISSN2041-6520
DOIs
Publication statusPublished - 2024

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