Tracking metal-binding proteins by fluorescence based approach

Abstract

Metal ions either essential or therapeutic play important roles in life processes or disease treatment. Proteins and enzymes are involved in metal homeostasis and action of many metallodrugs. Systematic identifications of metal or metallodrug binding proteins are particularly important towards understanding their roles in biology. The GE-ICP-MS1 developed by our group enables proteome-wide identification of proteins that bind metal ions strongly. Immobilized metal affinity column (IMAC) is also a useful method to mine putative metalloproteins from the complex proteomes, and to identify sequence motifs relating to the cellular functional behaviors of metals.2,3 However, it is of considerable challenges to track those proteins that bind to metallodrugs or metals weakly even transiently in live cells. New methodology is necessary for this purpose. We have designed and synthesized a family of metal-chelation (e.g. Ni-NTA) based fluorescent probes,4,5 consisting of a metal-NTA moiety, a fluorophore including coumarin, fluorescein, BODIPY and an arylazide. The probes can rapidly enter live cells to selectively label intracellular His6-tag proteins with significant fluorescence turn-on. The probe was further developed as metal-tunable fluorescence probes, allowing endogenous metal binding proteins to be labelled inside live cells and subsequently identified upon photo-activation of the arylazide. Using this approach, we have identified various metallo-proteomes including Bi(III), Fe(III), Cu(II) and Ni(II) proteomes in various prokaryotic and eukaryotic cells. Subsequent bioinformatics analysis revealed that the metal ions are enriched in various biological pathways.6 Using a similar strategy, we have developed an organoarsenic probe via conjugation of arsenic moiety with a fluorophore and arylazide, allowing downstream identification of arsenic binding protein in both NB4 and HL60 cells. This provide a basis for further analysis of mechanism of action of arsenic drugs. The methodology we describe here together with other (metallo)proteomics approaches will further advance our knowledge on the role of metals in biology and medicine.