Protein Stability Across Length and Time Scales

Doctoral thesis, 2026

Proteins are fundamental to all living organisms, and their correct folding is vital for many biological processes. A group of diseases collectively known as amyloidosis is characterised by the formation of amyloid fibrils, highly ordered protein aggregates that can accumulate in various tissues. Disaccharides, particularly trehalose, are known to stabilise proteins partly by elevating their denaturation temperature, thereby reducing unfolding and aggregation, yet the underlying mechanisms remain incompletely understood. Improving our understanding of protein misfolding and the role of sugars in
inhibiting amyloid fibril formation is therefore of great importance.
This thesis investigates how the disaccharides trehalose and sucrose influence protein stability, dynamics, and aggregation through their interactions with proteins and the surrounding aqueous environment. Neutron and X-ray scattering show that proteins remain preferentially hydrated in the presence of both sugars, with little direct interaction with the surface of the protein. Despite their structural similarity, trehalose reduces the protein dynamics to a greater extent than sucrose, consistent with a more pronounced coupling to the hydration water. Differential scanning calorimetry demonstrates that
both disaccharides enhance protein thermal stability by increasing the denaturation temperature.
In addition to stabilisation of the native state, the role of disaccharides in protein aggregation is examined. Small- and wide-angle X-ray scattering show that both trehalose and sucrose suppress aggregation and inhibit the formation of mature fibrils. Amyloid fibrils are commonly described as highly ordered and rigid structures. However, neutron spin echo and dielectric spectroscopy reveal that lysozyme amyloid fibrils retain internal dynamics similar in strength and time scale to those of the native protein, and that these dynamics, as in the monomers, are governed by the surrounding solvent.
Furthermore, the fibrils display flexible, polymer-like segmental dynamics in solution, revealing a pronounced multiscale dynamical behaviour, which may be important for understanding their role in neurodegenerative diseases.