Modulation of insulin aggregation by betaine and proline directly observed via real‐time super‐resolution microscopy

Protein aggregation is associated with a spectrum of neurodegenerative diseases. Although many small ligands have been found to modulate or inhibit protein aggregation, their molecular mechanisms remain unclear. One reason for this is the inherent heterogeneity of protein aggregation pathways with different kinetics that result in the coexistence of multiple structures, for example, protein spherulites and fibrils, challenging the analysis of protein-ligand interactions. To address this issue, we evaluated the roles of betaine and proline in insulin aggregation. We employed our recently developed super-resolution microscopy real-time kinetics via binding and photobleaching localization microscopy (REPLOM) to directly observe the formation and morphological evolution of individual insulin aggregates in real time, with or without betaine/proline. Utilizing our machine learning approach, we monitor the effect of betaine and proline on the aggregation pathways and extract the growth kinetics of each individual aggregate type. Our results show that a high concentration of betaine or proline modulates the heterogeneity of the final aggregates, leading to the formation of smaller aggregates in a mixture with spherulites. The fraction of small aggregates increases with betaine/proline concentration, highlighting the heterogeneity of protein aggregation, and our toolbox can unravel the effects of small molecule ligands on individual protein aggregation pathways and the resulting aggregate types and abundances.