Monitoring Early-Stage Protein Aggregation by an Aggregation-Induced Emission Fluorogen

Highly ordered protein aggregates, termed amyloid fibrils, are associated with a broad range of diseases, many of which are neurodegenerative, for example, Alzheimer's and Parkinson's. The transition from soluble, functional protein into insoluble amyloid fibril occurs via a complex process involving the initial generation of highly dynamic early stage aggregates or prefibrillar species. Amyloid probes, for example, thioflavin T and Congo red, have been used for decades as the gold standard for detecting amyloid fibrils in solution and tissue sections. However, these well-established dyes do not detect the presence of prefibrillar species formed during the early stages of protein aggregation. Prefibillar species have been proposed to play a key role in the cytotoxicity of amyloid fibrils and the pathogenesis of neurodegenerative diseases. Herein, we report a novel fluorescent dye (bis(triphenylphosphonium) tetraphenylethene (TPE-TPP)) with aggregation-induced emission characteristics for monitoring the aggregation process of amyloid fibrils. An increase in TPE-TPP fluorescence intensity is observed only with ordered protein aggregation, such as amyloid fibril formation, and not with stable molten globules states or amorphously aggregating species. Importantly, TPE-TPP can detect the presence of prefibrillar species formed early during fibril formation. TPE-TPP exhibits a distinctive spectral shift in the presence of prefibrillar species, indicating a unique structural feature of these intermediates. Using fluorescence polarization, which reflects the mobility of the emitting entity, the specific oligomeric pathways undertaken by various proteins during fibrillation could be discerned. Furthermore, we demonstrate the broad applicability of TPE-TPP to monitor amyloid fibril aggregation, including under diverse conditions such as at acidic pH and elevated temperature, or in the presence of amyloid inhibitors.