α-Synuclein is an abundantly expressed neuronal protein that contributes to a host of neurological conditions characterised by Lewy body formations, including Parkinson’s and dementia. α-Synuclein aggregation results in insoluble, beta-sheet rich amyloid fibrils, the growth of which is of key interest in studying the development of these diseases.
Seeded aggregation of α-synuclein monomer solutions was initiated in MES and PB buffers at pH 5.6 and 6.5 respectively using sonicated α-synuclein fibrils. Fibril growth in this solution was measured with Microfluidic Diffusional Sizing (MDS) using the FluidityOne and with ThT (Thioflavin T) spectroscopy using a Clariostar plate reader. The resulting size measurements from the Fluidity One and the change in β-sheet quantity from the ThT assay were normalised and plotted against time to map amyloid fibril growth.
The curves plotted from both data sets are highly comparable indicating MDS data represents a viable alternative to ThT spectroscopy for the measurement of α-synuclein amyloid fibril growth. However, MDS provides some advantages over ThT spectroscopy for this application. The ThT label is bound to the β-sheets of the fibril throughout the experiment and may affect the behaviour of the protein during measurement. MDS measures the protein in solution, in its native state and labelling only occurs following diffusion, preventing the label from influencing the measurement. Furthermore, some amyloid fibrils are not detected by ThT fluorescence and can provide false negative results. MDS has the capability to detect these fibrils due to its latent labelling system and direct detection of the size of protein species.
MDS represents a new approach for studying aggregation of α-synuclein and other proteins in their native state. Further to this, MDS bypasses many of the limitations of alternative techniques, such as requirement for surface functionalisation (SPR) or matrix separation (SEC), providing a new experimental option for researchers.