Bridging Mechanical Properties with Atomic Structures of Polymorphic α-Synuclein Fibrils by Single-Molecule Analysis and Molecular Dynamics Simulations

α-Synuclein (α-syn) forms structurally distinct fibril polymorphs with various pathological activities in different subtypes of synucleinopathies, such as Parkinson's disease (PD). As a unique proteinaceous polymer, the mechanical property of α-syn fibril is a primary determinant of its neurotoxicity, immunogenicity, and seeding and transmission capacity. Nevertheless, how genetic mutations in α-syn fibrils cause varied polymer behaviors remains largely unknown. Using optical tweezers, we quantitatively characterize the mechanical properties of three α-syn fibril variants at the single-molecule level. We find that wild-type α-syn fibrils are generally more sustainable to an axial disruption force than those formed by the disease-causing E46K and A53T α-syn mutants, whereas their heterogeneous elastic properties manifest similarity. Based on the molecular dynamics simulations, the β-sheet motif and the interface between the two protofilaments dominate in stabilizing the fibril structure. Additionally, single-molecule and simulation analysis consistently reveal the force-driven α-syn protein unfolding without a fibril break. Due to the flexible periphery, these subtle structural changes become more pronounced with the E46K fibril. The structure–mechanics relationship of α-syn fibrils built in this work sheds new light on the fibril assembly and disassembly mechanism and the mutant-associated pathogenesis in PD.