Precise Quantification of Molybdate In Vitro by the FRET-Based Nanosensor ‘MolyProbe’

Abstract

Molybdenum (Mo) is an essential trace element in all kingdoms of life. Mo is bioavailable as the oxyanion molybdate and gains biological activity in eukaryotes when bound to molybdopterin, forming the molybdenum cofactor. The imbalance of molybdate homeostasis results in growth deficiencies or toxic symptoms within plants, fungi and animals. Recently, fluorescence resonance energy transfer (FRET) methods have emerged, monitoring cellular and subcellular molybdate distribution dynamics using a genetically encoded molybdate-specific FRET nanosensor, named MolyProbe. Here, we show that the MolyProbe system is a fast and reliable in vitro assay for quantitative molybdate determination. We added a Strep-TagII affinity tag to the MolyProbe protein for quick and easy purification. This MolyProbe is highly stable, resistant to freezing and can be stored for several weeks at 4 °C. Furthermore, the molybdate sensitivity of the assay peaked at low nM levels. Additionally, The MolyProbe was applied in vitro for quantitative molybdate determination in cell extracts of the plant Arabidopsis thaliana, the fungus Neurospora crassa and the yeast Saccharomyces cerevisiae. Our results show the functionality of the Arabidopsis thaliana molybdate transporter MOT1.1 and indicate that FRET-based molybdate detection is an excellent tool for measuring bioavailable Mo.

Fluorescent Protein-Based Sensors for Detecting Essential Metal Ions across the Tree of Life

“…Recent efforts using MolyProbe have led to in vitro quantification of molybdate in extracts of plants and fungi. 209 ■ CURRENT CHALLENGES AND FUTURE…”

“…The fast response enabled transient changes in subcellular distributions of molybdate to be observed. Recent efforts using MolyProbe have led to in vitro quantification of molybdate in extracts of plants and fungi …”

Monitoring nutrients in plants with genetically encoded sensors: achievements and perspectives

“…This method cannot distinguish between free and protein-bound elements and lacks cellular and subcellular resolution. One of the first Mo sensors, MolyProbe, was composed of the FRET FP pair CFP and YFP and the bacterial-responsive protein ModE ( Oliphant et al 2022 ). MolyProbe responds to Mo in the nanomolar range ( K d = 4.7 × 10 −8 M) ( Anderson et al 1997 ; Nakanishi et al 2013 ), appropriate for intracellular measurements.…”

“…However, MolyProbe also reacts to sulfate ( K d = 2.7 × 10 −2 M) ( Nakanishi et al 2013 ). Latest efforts have led to in vitro quantifications of Mo in cell extracts of Arabidopsis and the fungi Neurospora crassa and Saccharomyces cerevisiae ( Oliphant et al 2022 ).…”

Tracing the Element: The Molecular Bases of Molybdenum Homeostasis in Legumes

“…In addition to the nonspecific pathway of molybdenum uptake into plants, two families of molybdate-specific transporters, MOT1 and MOT2 [21,73], have been identified in eukaryotes that ensure molybdate enters the cell through the plasma membrane. The MOT1 transporter gene is found only in the genomes of algae, mosses, and higher plants.…”