Protein misfolding and aggregation have been associated with a growing number of human diseases. As a multi-step process, protein aggregation involves formation of soluble misfolded oligomers and insoluble aggregates. The current method to monitor protein aggregation in live cells is through imaging fluorescent protein (FP)-tagged or fluorescently-labeled proteins, by analyzing changes in their location before and after aggregation. However, this method does not reveal whether proteins form misfolded soluble oligomers, which have been hypothesized to be the toxic species during protein aggregation. Thus, chemical tool that can visualize this process in live cells would advance our understanding of mechanism of these diseases. Herein, we present the application of structurally modulated FP chromophores, as synthetic small molecules, to selectively visualize misfolding and aggregation of a specific protein in live cells using turn-on fluorescence. Combining photophysical analysis, X-ray crystallography, and theoretical calculation, we show that fluorescence is triggered by inhibition of twisted-intramolecular charge transfer of these fluorophores in the rigid microenvironment in protein aggregates. Using purified proteins, we find that these FP-based probes can detect the previously invisible soluble oligomers during the process of protein aggregation. Based on these molecules, we further developed the AggTag (aggregation tag) method that employs bioorthogonal conjugation of the fluorophore to Halo-tag and SNAP-tag fused proteins. Unlike other methods, the AggTag method allows for fluorogenic detection of both the previously misfolded oligomers and insoluble aggregates in live cells. In summary, we demonstrate that the AggTag method with novel chemical probes is capable of detecting misfolded soluble proteins in live cells, a conformation that is difficult to visualize using classic non-fluorogenic methods.

Detecting the multi-step protein aggregation process in live cells using the AggTag method

2019 Fall National Meeting

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