Advancing amyloid fibril investigation using flow-induced dispersion analysis
We are pleased to announce a pioneering development in the domain of amyloid fibril investigation using flow-induced dispersion analysis (FIDA). Research done by protein biophysics laboratory at DTU Bioengineering introduces a new methodology tailored for the precise quantification of thermodynamic stability. This method not only optimises resource allocation, with sample volumes of less than a microliter per data point, but also operates in a label-free manner, ensuring broad accessibility across research contexts.
The study by Azad Farzadfard, Antonin Kunka, Thomas Oliver Mason, Jacob Aunstrup Larsen, Rasmus Krogh Norrild, Elisa Torrescasana Domínguez, Dr. Soumik Ray, and Alexander K. Buell delves into the landscape of fibril polymorphs, unveiling hitherto unexplored stability differentials. These findings hold significant promise in reshaping general understanding of structural diversity within amyloid fibrils and its implications for the pathogenesis of neurodegenerative diseases. This method advancement underscores the pivotal role of thermodynamic stability as a cornerstone parameter in deciphering the complex interplay of factors governing amyloid fibril dynamics.
We encourage you to access the full preprint here.
Thermodynamic stability of amyloid fibrils
To determine the thermodynamic stability of amyloid fibrils, FIDA was applied to αSyn fibrils equilibrated in varying concentrations of urea. Monomer quantification was performed using various methods, and a global fitting approach was chosen for its reliability and efficiency. The results were validated against other techniques such as differential scanning fluorimetry (DSF), static light scattering (SLS), Thioflavin T fluorescence, and ultracentrifugation (UCF).
The results have shown that these traditional methods used to analyze fibrils, have limitations in terms of sensitivity, throughput, and accuracy. Simultaneously, the paper shows that FIDA leverages the advantages of speed, sensitivity, and minimal sample consumption. See table below.
The paper suggests that FIDA enables rapid, in situ separation of fibrils and monomers using remarkably small sample volumes, as little as 5 μL, and it achieves this separation within minutes. Additionally, the process can be automated, facilitating high-throughput analysis, which is particularly valuable for extensive studies. Importantly, FIDA does not only rely on fluorescence-based detection, making it applicable even to proteins lacking tryptophan residues. This versatility extends its utility across a broad range of proteins, increasing its appeal as a versatile tool for researchers in the field of amyloid fibrils.
Furthermore, this research has shown that FIDA goes beyond quantitative measurements, as it provides qualitative insights into fibril properties by generating fluorescence signals that correlate with fibril size and aggregation state. This is instrumental for researchers seeking to optimize solution conditions to favor the formation of well-defined individual fibrils—a prerequisite for subsequent structural analyses using techniques like Atomic Force Microscopy (AFM) or cryo-Electron Microscopy (cryo-EM).
How does Flow Induced Dispersion Analysis work?
FIDA utilises Taylor dispersion to determine particle size based on their diffusivity within a laminar flow regime, characterised by low Reynolds numbers (Re < 2000). In laminar flow, fluid moves in smooth, parallel layers without turbulence. The flow's central layers are the fastest, while the outermost layers near the capillary wall are immobile, creating a characteristic parabolic flow velocity profile.
FIDA relies on particles' diffusion behaviour to differentiate between diffusive and non-diffusive species. Small particles, like molecules and proteins, diffuse between layers and exhibit Gaussian concentration profiles. Their diffusion coefficients are determined, allowing the calculation of hydrodynamic radii (Rh) using the Stokes-Einstein equation. In contrast, larger particles (Rh ≥ 100 nm), such as liposomes or protein aggregates, cannot radially diffuse at the applied pressure during the short experimental timescale and remain within the same flow layer, causing an asymmetric distribution at the detector. Watch this video for a visual representation.
The study's key innovation lies in using this phenomenon to separate non-diffusive aggregates from diffusive monomers, enabling the determination of amyloid fibril stability. Numerical simulations, validated by experiments, showcase how diffusive and non-diffusive particles behave in FIDA, emphasising the effectiveness of this technique in distinguishing between them. This approach offers a powerful tool for studying fibril stability and holds significant promise in the field of amyloid fibril research.
The complex landscape of amyloid fibril polymorphism.
Multiple polymorphic forms of amyloid fibrils have been observed in both clinical samples and laboratory settings. These structural variations are influenced by the specific conditions under which fibrils assemble, including factors like pH, salt concentration, incubation temperature, and agitation. Experiments presented in the paper explore the impact of salt and pH variations on the thermodynamic stability of fibril polymorphs.
The study reveals that distinct dominant polymorphs are formed under different conditions, leading to morphological differences observable through Atomic Force Microscopy (AFM). Surprisingly, the thermodynamic stability of these polymorphs varies depending on the conditions under which they are assayed. This variation can be attributed to the interplay between kinetic and thermodynamic factors during fibril formation. High ionic strength conditions, such as those induced by salt, accelerate the kinetics of fibril formation, resulting in polymorphs driven primarily by kinetic factors. Conversely, under low ionic strength conditions, electrostatic repulsion forces slow the aggregation process, allowing polymorphs to form with enhanced thermodynamic stability.
The paper also discusses the influence of pH on fibril stability. Fibrils formed under acidic conditions demonstrate faster aggregation kinetics and slightly higher stability compared to those formed at neutral pH. However, when transferred to physiological pH conditions, these fibrils experience a significant decrease in stability, highlighting the pivotal role of pH-induced changes in charge state in influencing fibril stability.
For more, we strongly encourage you to access the full preprint under this link: https://chemrxiv.org/engage/chemrxiv/article-details/651d32b68bab5d20559fa41e
Do you have questions that remain unanswered or would like to know if FIDA is applicable in your research? We will be happy to answer, send a question to one of our Field Application Scientists: https://www.fidabio.com/contact-us/question-to-a-scientist
