Tailoring the Amphiphilicity of Fluorescent Protein Chromophores to Detect Intracellular Proteome Aggregation in Diverse Biological Samples

The formation of amorphous misfolded and aggregated proteinsisa hallmark of proteome stress in diseased cells. Given its lack ofdefined targeting sites, the rational design of intracellular proteomeaggregation sensors has been challenging. Herein, we modulate theamphiphilicity of fluorescent protein chromophores to enable selectivedetection of aggregated proteins in different biological samples,including recombinant proteins, stressed live cells, intoxicated mouseliver tissue, and human hepatocellular carcinoma tissue. By tuningthe number of hydroxyl groups, we optimize the selectivity of fluorescentprotein chromophores toward aggregated proteins in these biologicalsamples. In recombinant protein applications, the most hydrophobicP0 (cLogP = 5.28) offers the highest fold change (FC = 31.6), sensitivity(LLOD = 0.1 & mu;M), and brightness (& phi; = 0.20) upon bindingto aggregated proteins. In contrast, P4 of balanced amphiphilicity(cLogP = 2.32) is required for selective detection of proteome stressesin live cells. In mouse and human liver histology tissues, hydrophobicP1 exhibits the best performance in staining the aggregated proteome.Overall, the amphiphilicity of fluorescent chromophores governs thesensor's performance by matching the diverse nature of differentbiological samples. Together with common extracellular amyloid sensors(e.g., Thioflavin T), these sensors developed herein for intracellularamorphous aggregation complement the toolbox to study protein aggregation.