Recognition of Proteins by Metal Chelation-Based Fluorescent Probes in Cells

Abstract

Fluorescent probes such as thiol-reactive and Ni²⁺-nitrilotriacetate (NTA) based probes provide a powerful toolbox for real-time visualization of a protein and a proteome in living cells. Herein, we first went through basic principles and applications of thiol-reactive based probes in protein imaging and recognition. We then summarize a family of metal-NTA based fluorescence probes in the visualization of His₆-tagged protein and identification of metalloproteins at proteome-wide scale. The pros and cons of the probes, as well as ways to optimize them, are discussed.

Keywords: His-tag; metallomics; metalloproteomics; molecular imaging; thiol-reaction.

Figure 1

The As(III)-based thiol-reactive fluorescent probes. (A) The recognition mechanism of As(III) binding toward two closely spaced cysteines. The buffer action can make several RAs(OH)₂ molecules to be protonated at the hydroxyl oxygen (R stands for any other chemical group). The attack of a thiolate anion on As(III) can release a water molecule. Then a proton transfer between the other thiolate anion and the hydroxyl oxygen is induced by the buffer action whilst the attack of this thiolate anion on As(III) will proceed an intramolecular displacement of another water molecule. (B) The structures of typical As(III)-based probes designed by different strategies. (A) was adapted from Gasser (2014) Copyright 2014 Wiley.

Figure 2

The probe family based upon NTA-metal coordination with their applications in protein tracking. Typical Ni-NTA-based probes were presented in (A) whilst our three generations of homemade probes were listed as (B), including Ni-NTA-AC, Ni-NTA-AF, and Ni-NTA-AB. Then the explorations for metal-associated proteomes in diverse system by using M-TRACER were presented as (C) the covalent labeling ability of M-TRACER toward intracellular proteins with UV-activation, (D) Fe-associated proteome tracked by Fe-TRACER in P. gingivalis and the protein-binding model released by X-ray crystallography, (E) Bi-associated proteome labeled by Bi-TRACER in H. pylori. (C,D) were reproduced from Jiang et al. (2018) with the permission of The Royal Society of Chemistry. (E) was reproduced from Wang et al. (2017) with the permission of The Royal Society of Chemistry.