van de Weert, M. | Search bioRxiv

bioRxiv2022

Heterogeneous and Surface-Catalyzed Amyloid Aggregation Monitored by Spatially Resolved Fluorescence and Single Molecule Microscopy

Amyloid aggregation is associated with many diseases and may also occur in therapeutic protein formulations. Addition of co-solutes is a key strategy to modulate the stability of proteins in pharmaceutical formulations and select inhibitors for drug design in the context of diseases. However, the heterogeneous nature of this multi-component system in terms of structures and mechanisms poses a number of challenges for the analysis of the chemical reaction. Combining a spatially resolved fluorescence approach with single molecule microscopy and machine learning approaches, we disentangle the different contributions from multiple species within a single aggregation experiment. Moreover, we link the presence of interfaces to the degree of heterogeneity of the aggregation kinetics and retrieve the rate constants and underlying mechanisms for single aggregation events, providing a general tool for a comprehensive analysis of self-assembly reactions. Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/510935v2_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@c678eaorg.highwire.dtl.DTLVardef@2c6decorg.highwire.dtl.DTLVardef@e4c2e4org.highwire.dtl.DTLVardef@b741aa_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

Polysorbate 80 Controls Morphology, Structure and Stability of Human Insulin Amyloid-Like Spherulites

Amyloid protein aggregates are not only associated with neurodegenerative diseases and may also occur as unwanted by-products in protein-based therapeutics. Surfactants are often employed to stabilize protein formulations and reduce the risk of aggregation. However, surfactants alter protein-protein interactions and may thus modulate the physicochemical characteristics of any aggregates formed. Human insulin aggregation was induced at low pH in the presence of varying concentrations of the surfactant polysorbate 80. Various spectroscopic and imaging methods were used to study the aggregation kinetics, as well as structure and morphology of the formed aggregates. Molecular dynamics simulations were employed to investigate the initial interaction between the surfactant and insulin. Addition of polysorbate 80 slowed down, but did not prevent, aggregation of insulin. Amyloid spherulites formed under all conditions, with a higher content of intermolecular beta-sheets in the presence of the surfactant above its critical micelle concentration. In addition, a denser packing was observed, leading to a more stable aggregate. Molecular dynamics simulations suggested a tendency for insulin to form dimers in the presence of the surfactant, indicating a change in protein-protein interactions. It is thus shown that surfactants not only alter aggregation kinetics, but also affect physicochemical properties of any aggregates formed.

biophysics↗