Probing the Energy Landscape of α-Synuclein Amyloid Fibril Formation by Systematic K-to-Q Mutagenesis

Aggregation of α-synuclein (αSyn) is a hallmark of Parkinson's disease, yet its pathological roles remain poorly understood. Elucidating these roles requires detailed knowledge of the free energy landscape of αSyn self-assembly, i.e. the states that can be populated and their mechanisms and rates of interconversion. Here, we quantitatively probe this landscape using systematic mutational analysis, focusing on electrostatic contributions. We designed and produced 41 αSyn variants containing one to six acetyllysine-mimicking (K-Q) mutations to dissect the effects on key assembly pathways, fibril stability, and polymorphism. We derived a quantitative framework for the analysis of mutational effects and used it to isolate general electrostatic from residue-specific effects on de novo αSyn aggregation. We established fibril stability and relative growth rate analysis as indirect biophysical probes of fibril polymorphism and identified K43Q+K45Q and K80Q as mutations with the most significant effect onamyloid formation. Moreover, we demonstrate that a subset of variants, most noticeably K58Q+K60Q, show differential sensitivity toward disease-derived fibril polymorphs in seed amplification assays. Clustering analysis reveals that the mutational effects map onto the structural context of the lysine residues in known αSyn fibril structures. Together, this work provides a scalable, quantitative framework for probing the complex αSyn assembly landscape using mutational analysis.