Protein Organisation at Different Scales: Amyloid Disaggregation, Chaperone Networks and Engineered Allostery

Living cells operate as thermodynamically open systems, perpetually sustained far from equilibrium by the continuous dissipation of energy. Among the most striking manifestations of this principle is proteostasis: the dynamic maintenance of the cellular proteome through a network of folding, disassembly, and degradation processes. Central to the proteostasis network are molecular chaperones, whose energy-consuming activities include enabling proteins to adopt and maintain functional conformations, and whose dysfunction is implicated in ageing and neurodegenerative disease. This thesis employs a biophysical, non-equilibrium thermodynamics perspective to comprehensively investigate chaperone systems and their activity in preventing and recovering from aggregation. The work is structured around four core studies, each probing different layers of biological complexity and spatial scale. We begin with a collaborative study on the disassembly of a-synuclein amyloid fibrils by the Hsp70 machinery (Hsc70, DNAJB1, Apg2), combining Thioflavin-T assays, electron microscopy, and mathematical modelling. The model confirms the widely observed phenomenon that disaggregation predominantly occurs at fibril tips, but also highlights the possibility that fragmentation plays a secondary, length-dependent role. This dual mechanism leaves distinct kinetic signatures, offering practical tools for distinguishing disassembly modes across amyloid systems. Zooming in, the next study develops an analytical and stochastic simulation framework for modelling Hsp70 binding to amyloid fibrils. We show that steric interactions are key to reproducing the experimentally observed clustering of Hsp70 at fibril tips, which, in turn, promotes depolymerisation. When combined with JDP-mediated ATP hydrolysis and NEF-driven nucleotide exchange, the model reveals the importance of energy-powered ultra-affinity, the central role of ATP-cycle dynamics, and the NEF's plausible multifaceted contributions. We then turn to small heat shock proteins, traditionally viewed as passive, ATP-independent holdases. By incorporating oligomerisation, substrate binding, and phosphorylation/dephosphorylation cycles into a reaction network model, we challenge this paradigm, showing that energy-driven regulation can fundamentally enhance sHsp function. This suggests that sHsps, like Hsp70, may also exploit flux-based mechanisms to achieve functionality inaccessible at equilibrium. Finally, inspired by natural allosteric transitions, we propose a design for a synthetic allosteric logic gate. Using elastic-network models and evolutionary optimisation, we construct a system that produces highly non-linear allosteric responses and performs logic-like operations. This programmable, energy-responsive network performs Boolean operations and paves the way for the engineering of synthetic molecular machines. Together, these studies build a coherent picture of energy-driven processes as the core of chaperone-mediated p