Proximity-Induced Covalent Labeling of Proteins with a Reactive Fluorophore-Binding Peptide Tag

Abstract

Labeling of proteins with fluorescent dyes in live cells enables the investigation of their roles in biological systems by fluorescence microscopy. Because the labeling procedure should not disturb the native function of the protein of interest, it is of high importance to find the optimum labeling method for the problem to be studied. Here, we developed a rapid one-step method to covalently and site-specifically label proteins with a TexasRed fluorophore in vitro and in live bacteria. To this end, a genetically encodable TexasRed fluorophore-binding peptide (TR512) was converted into a reactive tag (ReacTR) by adjoining a cysteine residue which rapidly reacts with N-α-chloroacetamide-conjugated TexasRed fluorophore owing to the proximity effect; ReacTR tag first binds to the TexasRed fluorophore and this interaction brings the nucleophilic cysteine and the electrophilic N-α-chloroacetamide groups in close proximity. Our method has several advantages over existing methods: (i) it utilizes a peptide tag much smaller than fluorescent proteins, the SNAP, CLIP, or HaLo tags; (ii) it allows for labeling of proteins with a small, photostable, red-emitting TexasRed fluorophore; (iii) the probe used is very easy to synthesize; (iv) no enzyme is required to transfer the fluorophore to the peptide tag; and (v) labeling yields a stable covalent product in a very fast reaction.

Quantifying lipid changes in various membrane compartments using lipid binding protein domains

“…When looking in the future we see better fluorescent proteins with higher quantum yield or improved photo-switchable features. Fluorescent tags that can be added to proteins using click chemistry such as the HALO, SNAP or CLIP tags have already been around and are used with great success [154–156] to offer an alternative to the larger fluorescent proteins. These would allow applications for superresolution imaging or moving toward high temporal resolutions.…”

Chemical Protein Modification through Cysteine

“…Alkylation of maltose‐binding protein (MBP) with a chloroacetamide‐appended fluorophore was achieved by Sunbul et al., followed by proximity‐induced binding to a fused fluorophore‐binding peptide. The methodology was found to achieve selective labelling of bacterial cells overexpressing the MBP‐fluorophore binding peptide fusion …”

On-Demand Targeting: Investigating Biology with Proximity-Directed Chemistry

“…Beyond CID-based implementation of tethered probes, 13 , 27 , 28 recent examples in the first subclass include proteasome targeting, 29 , 30 T cell activity control, 31 antibody recruitment, 9 , 32 and development of hybrid pharmaceuticals. 33 , 34 The second subclass is represented by multifunctional small-molecule-derived conjugates that function as potent and specific inhibitors, 35 , 36 biosensors, 37 , 38 reporters, 10 , 39 , 40 and protein-activity modulators. 41 , 42 …”