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

Area of Science:

• Biochemistry
• Molecular Biology
• Trace Element Analysis

Background:

• Molybdenum (Mo) is vital for life, functioning as the molybdenum cofactor in eukaryotes.
• Molybdate homeostasis is crucial, as imbalances cause deficiencies or toxicity.
• Fluorescence resonance energy transfer (FRET) methods, like MolyProbe, enable monitoring of cellular molybdate dynamics.

Purpose of the Study:

• To establish MolyProbe as a fast, reliable, and quantitative in vitro assay for molybdate determination.
• To demonstrate the utility of MolyProbe for measuring bioavailable Mo in various biological extracts.
• To validate the functionality of the Arabidopsis thaliana molybdate transporter MOT1.1 using the MolyProbe assay.

Main Methods:

• Genetically encoded molybdate-specific FRET nanosensor (MolyProbe) was engineered with a Strep-TagII for purification.
• MolyProbe system was optimized for stability, storage, and sensitivity, with peak performance at low nM molybdate levels.
• In vitro quantitative molybdate determination was performed on cell extracts from Arabidopsis thaliana, Neurospora crassa, and Saccharomyces cerevisiae.

Main Results:

• The MolyProbe system demonstrated high stability, resistance to freezing, and extended shelf-life at 4 °C.
• The assay exhibited optimal molybdate sensitivity at nanomolar concentrations.
• MolyProbe successfully quantified molybdate in plant, fungal, and yeast cell extracts, confirming MOT1.1 functionality in Arabidopsis thaliana.

Conclusions:

• The MolyProbe FRET nanosensor system is a robust and sensitive tool for in vitro molybdate quantification.
• This assay facilitates the measurement of bioavailable molybdenum across different life kingdoms.
• MolyProbe offers a valuable method for studying molybdate homeostasis and transporter function in biological systems.