First, I have designed and prepared (i) 76 aSyn variants containing one to eight K-to-Q mutations, (ii) 7 single-point E-to-Q mutants, (iii) 10 familiar mutants known to increase risk of the disease, and (iv) 30 variants carrying mutations in the regions forming the amyloid fibril cores. The 123 new aSyn variants represent an unprecedented number of sequence variation beyond the state-of-the-art, and is available upon request to the whole scientific community.
Furthermore, I have developed a medium-throughput expression-purification pipeline, that allowed me to prepare 30 mutant variants within 10 days in sufficient purity (>95% based on SDS-PAGE) and quantity (average of 1.7 mg) to perform all downstream assays. The protocol is flexible and easily transferable to any other research laboratory, increasing its impact beyond my research.
To study the effects of the mutations, I co-developed several experimental methods for characterizing aSyn self-assembly. First, the microfluidic transient incomplete separation used to analyze thermodynamic stability of amyloid fibrils from minute amounts of samples (Farzadfard and Kunka et al., 2024, Chemical Science). In the follow-up publication, I used the method to describe a novel quantitative link between thermodynamic stability and chaperone disaggregation (Fricke and Kunka, et al., 2025, under review in Chemical Science). Next, I co-developed new seed amplification assay that allows to amplify aSyn fibrillar material from the CSF or brain tissues of PD or MSA patients (Farzadfard 2025, Angewandte Chemie Int. Ed.). Finally, I created a new experimental protocol for studying amyloid formation of WT and familiar mutants of aSyn within a dense phase upon de-mixing of aSyn solution under crowding conditions (Ray and Kunka, et al., 2025 under review in Nature Communications).
I used the 76 K-to-Q aSyn mutants to study the effect of systematic positive charge removal on de novo aggregation, elongation, cross-seeding, fibril stability, secondary nucleation, liquid-liquid phase separation, lipid binding, and lipid-induced aggregation. I quantified the energetic changes induced by the mutations to the kinetics (energy barrier of the elongation) and thermodynamics (fibril stability) and identified mutations of lysines found in the protofibril interface of WT fibrils that play crucial role in aSyn polymorphism. The analysis is now being extended by the 30 conservative mutations that will allow to carry out the phi-value analysis, which was not applicable to the previous set due to the pronounced changes in polymorphism of the elongated products. For the KQ mutants, I found a negative correlation between the elongation rate and fibril stability, underlying the important roles of electrostatics in both kinetics and thermodynamics of aSyn assembly.
The correlation analysis of the mutational effects allowed me to cluster the microscopic pathways based on their sensitivity to mutations, identifying shared interaction networks between them. Apart from the one mentioned above, I found positive correlation between secondary nucleation and lipid-induced aggregation indicating similar conformations being sampled by the protein. In general, I found that the sequence position, rather than the number of mutations dictate the assembly behavior of aSyn in most of the assays. This was reflected in the poor correlation between experimental observations, and results of molecular dynamics (MD) simulations using state-of-the-art coarse-grained force-field CALVADOS2. Neither changes in compactness, prolateness, nor radius of gyration derived from the MD could explain the differences between the experimental aggregation behavior of the mutants. Together, this strongly suggest that specific local interactions, rather than global net charge play important role in self-assembly of aSyn.
Most of the studies to date focus on a few assays and a few mutants, and trying to correlate experimental observations with in vivo effects. The unprecedented number of mutants and assays probed within the project here revealed their weaknesses, and highlighted the importance of providing a much broader perspective on mutational effects in proteins with complex rugged landscapes such as aSyn. The results are summarized in two soon-to-be submitted first author manuscripts, two shared first-author publications, and two co-authored publications.
