Alpha-synuclein (α-synuclein) is a small protein highly expressed in the brain, playing a central role in the pathology of neurodegenerative diseases like Parkinson’s disease, Lewy body dementia, and multiple system atrophy. Its tendency to misfold and aggregate underlies the formation of toxic fibrils and Lewy bodies, hallmark features of these diseases. Understanding, and controlling, the alpha-synuclein aggregation process is critical for developing targeted therapies, but this protein presents several experimental challenges.
This blog outlines three major hurdles in characterizing alpha-synuclein aggregation and highlights the use of Microfluidic Diffusional Sizing to carry out the Neuroaffinity and Stoichiometry (NeSt) assay, which can help overcome these challenges.
Key Challenges in Studying Alpha-Synuclein Aggregation
1. Dynamic Behavior due to its Intrinsically Disordered Nature
Alpha-synuclein is an intrinsically disordered protein (IDP) [1]. It lacks a stable secondary or tertiary structure in its monomeric form, making it challenging to characterize using traditional structural biology techniques. Being an IDP means that alpha-synuclein has no fixed conformation but is conformationally adaptive/flexible. It is crucial to determine the properties and behavior of the biologically relevant state and not a proximity-induced form.
Adding to the challenge, alpha-synuclein’s behavior is highly sensitive to environmental factors [2]. Minor changes in temperature, pH, ionic strength, or even subtle variations in buffer composition can drastically affect its folding and aggregation kinetics. This sensitivity extends beyond steric effects, potentially creating bias and complicating experiments.
2. Polymorphism and Complex Stoichiometry
Alpha-synuclein aggregates can form a range of polymorphic fibril structures, depending on experimental or physiological conditions [3]. This polymorphism is not merely structural; it can influence fibril stability, toxicity, and seeding capability, leading to variability in experimental data. This diversity complicates understanding the exact mechanisms driving pathology and identifying reliable therapeutic targets.
Aggregates of alpha-synuclein often involve heterogeneous populations with varying stoichiometries. Studying these mixtures requires advanced tools capable of analyzing these complex assemblies without losing critical context. Determination of binding stoichiometry, therefore, is challenging for many traditional interaction analysis approaches.
3. Challenges with Size, Shape and Orientation
Aggregated alpha-synuclein species span a broad size range, from small oligomers to large fibrils. This variability complicates biophysical measurements. For instance, larger aggregates may be difficult to immobilize on a surface, confounding sample preparation and measurement, and leading to incomplete or skewed data.
When immobilization or binding assays are used, the orientation of alpha-synuclein aggregates becomes critical. If epitopes are buried or occluded, the assay may yield misleading results, compromising subsequent experimental conclusions and decision-making.
MDS/ NeSt as a Recommended Assay for Alpha-Synuclein Aggregation
Microfluidic Diffusional Sizing (MDS) is a recently developed solution-phase protein interaction technology that is well suited to studying proximity-sensitive systems such as IDPs. MDS leverages the well-established relationship between molecular size and diffusion rate to deliver absolute size measurements. When two proteins bind, the resulting complex is larger than the individual components, and MDS detects this size increase to reveal binding interactions.
Building upon MDS, the Neuroaffinity and Stoichiometry (NeSt) assay is a dedicated assay for determining both binding affinity and stoichiometry of interactions. Remarkably, NeSt achieves this without requiring external calibrants or prior knowledge of the interaction.
Key Advantages of NeSt for Alpha-Synuclein Aggregation Characterization
By measuring the hydrodynamic radius of proteins in solution, NeSt enables studying compactness or foldedness of alpha-synuclein aggregates with ease. It works directly in native environments, such as crude cell lysates or cerebrospinal fluid, without the need for immobilization or purification, overcoming the common hurdles caused by immobilizing alpha-synuclein on a surface and changing environmental conditions.
NeSt employs a fluorescently labeled probe protein to detect size increases when it binds to an unlabeled target protein. This design enables efficient determination of binding affinities even in heterogeneous mixtures.
Finally, NeSt simplifies stoichiometry analysis by integrating with the Fluidity Insight platform. This intelligent platform uses standardized models to efficiently calculate interaction stoichiometry, bypassing the need for external calibrants or reference standards.
Conclusion
Characterizing alpha-synuclein aggregation is critical for advancing our understanding of neurodegenerative diseases and developing effective therapies. However, its intrinsically disordered nature, sensitivity to environmental factors, polymorphic aggregation, and challenges related to size and orientation make it a particularly difficult target for traditional methods. Microfluidic Diffusional Sizing (MDS) technology and the Neuroaffinity and Stoichiometry (NeSt) assay offer a transformative approach to overcome these hurdles. By working directly in native environments without the need for immobilization or purification, MDS/NeSt ensures efficient and unbiased affinity measurement of alpha-synuclein aggregates, and allows study of their compactness, foldedness and stoichiometry, even in complex heterogeneous mixtures.
Follow the link below to learn more about MDS or contact us to discuss how MDS can assist your alpha-synuclein study.
References
- Bisi, Nicolò, et al. “α-Synuclein: an all-inclusive trip around its structure, influencing factors and applied techniques.” Frontiers in Chemistry 9 (2021): 666585. DOI: 10.3389/fchem.2021.666585
- Gatto, Nicole M., et al. “α-Synuclein gene may interact with environmental factors in increasing risk of Parkinson’s disease.” Neuroepidemiology 35.3 (2010): 191-195. DOI: 10.1159/000315157
- Mehra, Surabhi, et al. “Structural and functional insights into α-synuclein fibril polymorphism.” Biomolecules 11.10 (2021): 1419. DOI: 10.3390/biom11101419