Energetic profiling reveals thermodynamic principles underlying amyloid fibril maturation

Amyloid fibrils adopt diverse structural polymorphs linked to disease-specific phenotypes, but the thermodynamic principles guiding their formation and maturation remain unclear. Here, we apply energetic profiling to structural time series from cryo-EM datasets of IAPP, tau, and α-synuclein to decode the principles governing fibril maturation and polymorphic divergence. By mapping residue-level free energy contributions across experimentally resolved assembly pathways, we reconstruct their maturation trajectories and find that amyloid assembly is anchored by aggregation-prone regions that serve as sequence-encoded stabilizing motifs. As assembly progresses, these motifs are reorganized and expanded, while additional regions introduce structural frustration that enables conformational flexibility. Environmental cofactors such as metal ions or polyanions are observed in association with regions of structural remodeling, where they may act to compensate for otherwise energetically strained conformations. This framework offers mechanistic insight into how distinct polymorphs arise from a common sequence and mature through both intrinsic and extrinsic thermodynamic influences. Energetic profiling of cryo-EM time series for IAPP, tau, and α-synuclein reveals how amyloid fibril maturation is driven by sequence-encoded hotspots. Rearrangement of APRs anchors growth, while cofactors relieve strain, enabling polymorphic diversity.