Jacob Aunstrup Larsen

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.

Amyloid-forming peptides are increasingly recognized as dynamic regulators at the host–pathogen interface, yet how environmental factors control their assembly and activity remains poorly understood. Here, RNA acts as a concentration-dependent regulator of two sequence-related α-helical peptides with fundamentally different assembly behaviors: the cross-α amyloid-forming Staphylococcus aureus virulence factor PSMα3 and the non-amyloidogenic human host-defense peptide LL-37. RNA drives PSMα3 through distinct assembly states, from liquid-like condensates to fibrillar polymorphs, while preserving cytotoxic and antimicrobial activity over time. In contrast, RNA attenuates LL-37 cytotoxicity toward host cells while maintaining antibacterial activity, consistent with a host-protective immunomodulatory effect. Together with the opposing effects of epigallocatechin gallate, which redirects both peptides into amorphous assemblies, these findings support a mechanistic model in which biological activity is governed by supramolecular architecture, assembly trajectory, and dynamics rather than by monomer abundance or mature fibrils alone. More broadly, our findings identify RNA as an environmental regulator of α-helical peptide assemblies, and establish assembly-state control as a tunable determinant of virulence and host defense.

Therapeutic peptides are emerging as an important class of pharmaceutical compounds able to bridge the gap between small molecules and large biologics. Although modern peptide synthesis protocols have significantly increased the throughput of drug discovery, developability assessment remains both material- and labour-intensive, creating a bottleneck in development pipelines. Here we present an automated micro-capillary solubility assay able to quantify peptide solubility using only a few µg of product from a single stock solution of either aqueous or DMSO-solubilized peptide, thereby minimising laborious sample preparation. We demonstrate its utility with two families of therapeutic peptides: dual GLP1/glucagon agonists and Y2 peptide agonists. Beyond solubility assessment, the methodology generates additional data on self-association, stability in ionic solutions and identifies glass-surface binding compounds. Combined, this information allows to identify types of oligomerization behaviour both within and across the peptide families, rationalizing differences in solubility and biophysical properties. This platform therefore represents a valuable novel tool in early screening and characterization.

The aggregation of natively disordered α-Synuclein (αSyn) into amyloid fibrils is a hallmark of Parkinson’s and other neurodegenerative diseases. Understanding αSyn’s pathological role remains a major challenge due to its complex, context-dependent energy landscape characterized by conformational plasticity and fibril polymorphism. Here, we present a systematic mutational analysis as a quantitative probe of the αSyn energy landscape, focusing on electrostatic contributions to key aggregation pathways. We engineered αSyn variants with one to eight lysine-to-glutamine substitutions and analyzed their aggregation under controlled conditions to delineate their effects on nucleation, elongation, seed amplification, fibril stability, and fibril polymorphism. We find that αSyn aggregation from a homogenous solution can be modelled well using global properties, including protein concentration, charge, and ionic strength. Microscopic pathways and the resulting fibril polymorphs are instead modulated by sequence-specific effects. We identify mutations of residues found in fibril cores as perturbations that significantly modify the αSyn free energy landscape, creating pathways and energy minima not accessible to the WT under the same experimental conditions. In contrast, mutations outside of the fibril core affect the magnitude of the relevant energy barriers whilst overall maintaining a WT-like free energy landscape. Our work outlines a scalable, quantitative framework that increases the informational output of the mutational studies of αSyn using conventional assays. The approach can be extended by incorporating additional mutational and functional data to deepen our understanding of αSyn’s energy landscape and its role in health and disease.

We study the role of the fuzzy coat of α-synuclein amyloid fibrils via proteolytic degradation. Bulk solution assays offer limited insight, while Quartz crystal microbalance can reveal detailed mechanisms of growth of the “shaven” amyloid fibrils.

Amyloid fibrils are highly stable misfolded protein assemblies that play an important role in several neurodegenerative and systemic diseases. Although structural information of the amyloid state is now abundant, mechanistic details about the misfolding process remain elusive. Inspired by the Φ-value analysis of protein folding, we combined experiments and molecular simulations to resolve amino-acid contacts and determine the structure of the transition-state ensemble—the rate-limiting step—for fibril elongation of PI3K-SH3 amyloid fibrils. The ensemble was validated experimentally by Tanford β analysis and computationally by free energy calculations. Although protein folding proceeds on funnel-shaped landscapes, here we find that the energy landscape for the misfolding reaction consists of a large ‘golf course’ region, defined by a single energy barrier and transition state, accessing a sharply funnelled region. Thus, misfolding occurs by rare, successful monomer–fibril end collisions interspersed by numerous unsuccessful binding attempts. Taken together, these insights provide a quantitative and highly resolved description of a protein misfolding reaction.

Amyloid fibrils typically consist of a dense core made up of β-strands, with disordered flanks on either side, and sometimes interrupted by disordered loop regions (the fuzzy coat). α-synuclein found in Lewy Bodies of Parkinson’s Disease patients are mostly C-terminally truncated, meaning that a large fraction of the fuzzy coat of disease-related fibrils is enzymatically degraded in the amyloid state. We demonstrate that the proteolytic removal of the fuzzy coat leads to enhanced fibril-fibril interactions and flocculation, which renders the study of the role of the fuzzy coat in bulk solution very challenging. In order to overcome these challenges, here we use Quartz Crystal Microbalance with Dissipation (QCM-D), a surface based biosensing technique, to study the effects of proteolytic removal of the fuzzy coat of α-synuclein amyloids. We demonstrate that DissipationFrequency analysis can illuminate multiple simultaneous reactions and characterize the monomer-fibril interactions in detail. We find that removal of the fuzzy coat increases apparent fibril elongation rates permanently upon each proteinase treatment. Utilizing kinetic models, we demonstrate that our results cannot be rationalized by alterations of the elongation rate of fibrils alone, but indicates that removal of the fuzzy coat of α-synuclein fibrils enable secondary processes at neutral pH. We propose that the nature of the secondary processes investigated here may be highly relevant for understanding disease-related α-synuclein amyloid formation. Furthermore we suggest that the Quartz Crystal Microbalance with Dissipation is a particularly attractive platform for studying the effects of post-translational modifications on amyloid growth and molecular interactions.

Amyloid fibrils are highly stable misfolded protein assemblies playing an important role in several neurodegenerative and systemic diseases. While structural information of the amyloid state is now abundant, mechanistic details about the misfolding process remain elusive. Here we present a Φ-value inspired approach and apply it to PI3K-SH3 amyloid fibrils to examine the rate-limiting step for fibril elongation. We use experimental Φvalues as constraints in biased MD-simulations to provide the first view of the transition state of a protein misfolding reaction. The resulting framework is generally applicaple and provides mechanistic insight into the misfolding reaction comparable to the breakthroughs previously achieved for protein folding. While protein folding proceeds on funnel-shaped landscapes, we find that the misfolding reaction energy landscape consists of a large ’golf course’ region, defined by a single energy barrier and transition state, accessing a sharply funneled region. Thus, misfolding occurs by numerous unsuccesful binding attempts and rare successful monomer-fibril end collisions which rapidly anneals to the final state. Taken together, these insights enable, the first quantitative and highly resolved description of a protein misfolding reaction.

Amyloid fibrils of proteins such as α-synuclein are a hallmark of neurodegenerative diseases and much research has focused on their kinetics and mechanisms of formation. The question as to the thermodynamic stability of such structures has received much less attention. Here, we newly utilize the principle of transient incomplete separation of species in laminar flow in combination with chemical depolymerization for the quantification of amyloid fibril stability. The relative concentrations of fibrils and monomer at equilibrium are determined through an in situ separation of these species based on their different diffusivity inside a microfluidic capillary. The method is highly sample economical, using much less than a microliter of sample per data point and its only requirement is the presence of aromatic residues (W, Y) because of its label-free nature, which makes it widely applicable. Using this method, we investigate the differences in thermodynamic stability between different fibril polymorphs of α-synuclein and quantify these differences for the first time. Importantly, we show that fibril formation can be under kinetic or thermodynamic control and that a change in solution conditions can both stabilise and destabilise amyloid fibrils. Taken together, our results establish the thermodynamic stability as a well-defined and key parameter that can contribute towards a better understanding of the physiological roles of amyloid fibril polymorphism.

Protein liquid–liquid phase separation can lead to disease-related amyloid fibril formation. The mechanisms of conversion of monomeric protein into condensate droplets and of the latter into fibrils remain elusive. Here, using mass photometry, we demonstrate that the Parkinson’s disease-related protein, α-synuclein, can form dynamic nanoscale clusters at physiologically relevant, sub-saturated concentrations. Nanoclusters nucleate in bulk solution and promote amyloid fibril formation of the dilute-phase monomers upon ageing. Their formation is instantaneous, even under conditions where macroscopic assemblies appear only after several days. The slow growth of the nanoclusters can be attributed to a kinetic barrier, probably due to an interfacial penalty from the charged C terminus of α-synuclein. Our findings reveal that α-synuclein phase separation occurs at much wider ranges of solution conditions than reported so far. Importantly, we establish mass photometry as a promising methodology to detect and quantify nanoscale precursors of phase separation. We also demonstrate its general applicability by probing the existence of nanoclusters of a non-amyloidogenic protein, Ddx4n1.