Individual filament structural analysis of peptide amyloid assemblies containing D-amino acids

Amyloids represent a class of insoluble, fibrillar, protein structures with a tendency to aggregate and a heavy association with neurodegenerative diseases. The segment sequence VQIVYK, also referred to as PHF6, from the tau protein associated with Alzheimer’s disease and tauopathies is crucial in the aggregation process associated with these disorders. The hexapeptide is capable of forming amyloid fibrils in vitro comprised of the characteristic paired β-sheets whose variation in arrangement results in polymorphs. Polymorphism is a key challenge in amyloid research and therapeutic development, as structural heterogeneity can arise even when identical conditions and building blocks are used. Chirality plays a pivotal role in peptide formation and self-assembly, significantly influencing the resulting structures. This study investigates how a single amino acid substitution (valine to tryptophan) and chirality (L- vs. D-) in VQIVYK analogues affect fibril assembly, kinetics, and polymorphism. Tryptophan’s bulk and aromaticity introduce new interactions, impacting stability and revealing the roles of aromaticity and steric effects in aggregation. In vitro fibril formation occurred in Phosphate buffer, static incubation at 25°C, initial assembly was monitored after two weeks. Using atomic force microscopy (AFM), we imaged and generated the 3D structural reconstructions of individual filaments formed by these peptides. The results show that residue chirality can have profound effects on the long-range structural features of peptide/protein assemblies, exhibiting chiral control during amyloid formation and influencing the morphology of resulting aggregates. Quantitative analysis of the extracted AFM data showed these isomeric structures result in a mirrored inherent polymorphic distribution, suggesting chirality presides the self-assembly pathway. The insights gained from studying self-assembly and the impact of D-peptides on fibril formation, hold promise for advancing both therapeutics and biotechnology. Understanding D-amino acids’ influence fibril formation could provide novel strategies for controlling or disrupting pathological aggregation in neurodegenerative diseases. Additionally, manipulating fibril formation via D-amino acids enables the design of chiral biomaterials and nanostructures with tuneable self-assembly.