Modulation of Fluorescent Protein Chromophores To Detect Protein Aggregation with Turn-On Fluorescence

Abstract

We present a fluorogenic method to visualize misfolding and aggregation of a specific protein-of-interest in live cells using structurally modulated fluorescent protein chromophores. Combining photo-physical 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 of viscous solvent or protein aggregates. Bioorthogonal conjugation of the fluorophore to Halo-tag fused protein-of-interests allows for fluorogenic detection of both misfolded and aggregated species in live cells. Unlike other methods, our method is capable of detecting previously invisible misfolded soluble proteins. This work provides the first application of fluorescent protein chromophores to detect protein conformational collapse in live cells.

A Fluorogenic AggTag Method Based on Halo‐ and SNAP‐Tags to Simultaneously Detect Aggregation of Two Proteins in Live Cells

“…On the other hand, bright perinuclear, punctate fluorescence of P2 was observed in cells expressing Htt‐Q97 ⋅ SNAPf (Figure B, bottom). This fluorogenic pattern is consistent with our previous finding that P1 detects aggregates through punctate fluorescence in cells expressing Htt‐Q97 ⋅ Halo …”

“…The previously developed AggTag probe P1 contains a Halo‐tag ligand and a synthetic fluorescent protein chromophore ( λ ex =530 nm, λ em =630 nm, Scheme A) . To develop a new AggTag probe useable for SNAP‐tag fusion proteins, we designed P2 , based on a SNAP‐tag ligand and a 7‐dimethylamino‐4‐sulfamonyl‐2,1,3‐benzothiadiazole (SBD, λ ex =445 nm, λ em =570 nm) moiety as a solvatochromic fluorophore (Scheme B).…”

“…Synthesis of the AggTag probes : The AggTag probes P1 and P3 , based on the Halo‐tag ligand, were synthesized according to the previously reported procedure . The AggTag probes P2 and P4 , based on the SNAP‐tag ligand, were synthesized by using compounds 1 a and 1 b as synthetic precursors; these in turn were also prepared according to a previously reported procedure (Scheme S1) …”

“…To address this limitation, fluorogenic (turn‐on fluorescence) detection could be a powerful means to detect biological processes with minimal background . Along these lines, we recently developed a new imaging method named AggTag ( Agg regation Tag ) to detect soluble oligomers and insoluble aggregates by turning on fluorescence in live cells (Scheme A) . In this method, a Halo‐tag is fused to a POI and labeled with Halo‐tag ligand P1 , containing a fluorophore based on a molecular rotor.…”

A Maleimide‐functionalized Tetraphenylethene for Measuring and Imaging Unfolded Proteins in Cells

“…reported a conjugate of a chemical probe and a de novo designed enzyme, which formed fluorescent aggregates in the presence of cellular stress . A similar approach utilizing a fluorogenic probe targeting an aggregation‐prone “HaloTag” protein mutant has also been reported by the same group . Additionally, other methods that focus on cell lysate analysis have also been documented .…”