Covalent Solvatochromic Proteome Stress Sensor Based on the Schiff Base Reaction

Abstract

Covalent-type probes or sensors have been seldom reported for aggregated proteins. Herein, we reported a series of covalent solvatochromic probes to selectively modify and detect aggregated proteomes through the Schiff base reaction. Such covalent modification was discovered by serendipity using the P1 probe with an aldehyde functional group, exhibiting enhanced fluorescence intensity and unusually large blue shift upon protein aggregation. Supported by the biochemical and mass spectrometry results, we identified that this probe can modify the lysine residue of aggregated proteins selectively over folded ones via the Schiff base reaction. The generality of designing such a covalent-type probe was demonstrated in multiple probe scaffolds using different model proteins. Finally, we exploited the distinct solvatochromism of P1 after Schiff base linkage with aggregated proteins to visualize the distinct morphology of aggregated proteomes, as well as to quantify the polarity heterogeneity inside it. This work may intrigue the exploration of other chemical reaction types to covalently functionalize aggregated proteins that were difficult to analyze.

Developing an Affinity-Based Chemical Proteomics Method to In Situ Capture Amorphous Aggregated Proteome and Profile Its Heterogeneity in Stressed Cells

“…We next sought to covalently capture the aggregated proteins by anchoring reactive warheads on probe P1 and enabling proximity induced bioconjugation. We previously reported cysteine and lysine residues of the aggregated proteome can be covalently and selectively modified by aggregated proteome imaging sensors. , Encouraged by these findings, we further modulated the warheads’ reactivity to balance the selectivity and labeling kinetics. First, as the photoaffinity groups can globally label diverse amino acids via radical chemistries, we functionalized scaffold P1 with different photoaffinity groups, including benzophenone (P4), trifluoromethylphenyl diazirine (P5), and 3-hydroxy-2-naphthalenemethanol derivative (P6).…”

Fluorogenic methodology for visualization of phase separation in chemical biology

“…Ye 71 et al used the AggTag method to directly monitor protein oligomers, and could distinguish insoluble aggregates from other conformations in living cells. The internal polar heterogeneity of aggregates can also be quantified by a series of covalent solvent-changing probes that selectively modify and detect the aggregated proteome by Schiff base reactions 72 (Fig. 6b).…”

Design and Application of Fluorescent Probes to Detect Cellular Physical Microenvironments

“…This result demonstrated that NTPAN-MI (P61) not only reported on the unfolded protein located in the cytoplasm, but also, for the first time, in the nucleus. In 2022, Liu et al 109 reported another polarity sensitive probe, P5 (P62), with a reactive aldehyde moiety. Supported by the biochemical and mass spectrometry results, they have determined that the Schiff base reaction enabled this probe to selectively modify the lysine residue of aggregated proteins, instead of the folded ones.…”

“…So far, numerous polarity sensitive probes have been reported for these purposes. These works pave the way to a series of chemical strategies to design fluorescent polarity sensors for fine characterization of the heterogeneous polarity at multiple levels, from subcellular organelles like a lipid droplet or protein aggresome, , up to a living organism like the zebrafish and mouse disease model. , However, several challenges remain to be tackled by chemists when designing novel polarity sensors.…”