Structural Insights Into Amyloid Polymorphism: The Impact of Glutamine to Norleucine Substitutions in GNNQQNY Aggregation.

Polypeptides can self-assemble into highly organized amyloid structures through complex and poorly understood mechanisms. To better understand the key parameters governing amyloidogenesis, we investigated the aggregation of the Sup35 prion-derived GNNQQNY sequence alongside two rationally designed mutants, glutamine to norleucine in the 4th or 5th position, where selective removal of hydrogen bonding capacity reduces amyloid structural stability. Our findings reveal that β-sheet arrays form rapidly as an initial step, followed by π-π aromatic interactions between Tyr residues, which drive hierarchical self-assembly into 3D fibrillar structures via hydrophobic zippers and partial water exclusion. As the oligomers grow, they also acquire twist and chirality at the protofilament level, with Tyr ladders serving as key interaction surfaces that dictate the final amyloid architecture. These ladders guide protofibrils to assemble into either oppositely twisted chiral fibers or achiral nanocrystals, contributing to amyloid polymorphism. The emergence of distinct polymorphs is influenced by multiple factors, including fibril twisting, side-chain interactions, solvent exclusion, and local microenvironmental conditions. Our study provides crucial insights into the hierarchical nature of amyloid self-assembly and highlights the structural adaptability of amyloid fibrils, which is essential for designing functional amyloids and understanding the pathogenicity of disease-associated aggregates.