Abstract
Molybdenum (Mo) is an essential trace element for almost all living organisms including animals. Mo is used as a catalytic center of molybdo-enzymes for oxidation/reduction reactions of carbon, nitrogen, and sulfur metabolism. Whilst living cells are known to import inorganic molybdate oxyanion from the surrounding environment, the in vivo dynamics of cytosolic molybdate remain poorly understood as no appropriate indicator is available for this trace anion. We here describe a genetically encoded Förester-resonance-energy-transfer (FRET)-based nanosensor composed of CFP, YFP and the bacterial molybdate-sensor protein ModE. The nanosensor MolyProbe containing an optimized peptide-linker responded to nanomolar-range molybdate selectively, and increased YFP:CFP fluorescence intensity ratio by up to 109%. By introduction of the nanosensor, we have been able to successfully demonstrate the real-time dynamics of molybdate in living animal cells. Furthermore, time course analyses of the dynamics suggest that novel oxalate-sensitive- and sulfate-resistant- transporter(s) uptake molybdate in a model culture cell.
Highlights
Molybdenum (Mo) is an essential microelement for most living organisms, in both prokaryotes and eukaryotes [1,2]
Development of Molybdate Sensing Protein A genetically-encoded FRET nanosensor for molybdate was constructed by CFP-variant Cerulean [23], YFP-variant cp157Venus [24] and a molybdate binding domain (MoBD) (Figure 1A)
The intramolecular assembly of the two molybdate binding domains (MoBD) induced by molybdate was expected to reduce the distance between CFP and YFP, and/or narrow the solid angle between the two chromophores, increasing FRET efficiency (Figure 1B)
Summary
Molybdenum (Mo) is an essential microelement for most living organisms, in both prokaryotes and eukaryotes [1,2] It is utilized as a catalytic center in molybdoenzymes to oxidize and reduce carbon, nitrogen and sulfur metabolites, since this element is one of the transition metal elements with an oxidation state varying from +2 to +6. Most molybdoenzymes, such as xanthine oxidase, sulfite oxidase, aldehyde oxidase, nitrate reductase, and mitochondrial-amidoxime-reducing-component, contain a pterinbased Mo cofactor (Moco) [3,4], while a bacterial nitrogenase includes iron-Mo cofactor (FeMo-co). The dynamics of cytosolic molybdate and its related uptake systems are as yet unknown
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