Beyond the Amyloid Core: Modulation of Aggregate Conformational Diversity by Mutations in the Noncore Regions

Emerging evidence underscores the regulatory roles of nonamyloidogenic regions in controlling the aggregation dynamics and cytotoxicity of amyloidal proteins, but the mechanism remains unclear. Herein we investigated how flanking sequences modulate the conformational heterogeneity in the p53 238-262 amyloid segment using scanning tunneling microscopy (STM). By comparing the wild-type (wt) and three pathogenic mutations (R248W, R248Q, R249S) in the noncore regions, we reveal that flanking alterations remodel β-sheet aggregates and induce conformational plasticity in β-strand ensembles through the generation of novel conformational substates and selective elimination of existing conformational substates. This conformational diversity establishes a highly heterogeneous intermolecular interaction network that connects different conformational substates. Quantitative mapping shows that flanking mutations drastically diversify the coexisting inter-β-strand interactions while altering the most abundant intermolecular interaction patterns. Our analysis reveals that flanking mutations induce a deterministic conformational remodeling process in the β-sheet aggregates, where specific intermolecular interactions counteract entropy through nonstochastic structural rearrangements. These findings contribute to the mechanistic understanding of the noncore sequence control in protein aggregation and suggest potential therapeutic targets for modulating β-sheet assembly in amyloidopathies.