A Fluorescent, Reagentless Biosensor for ATP, Based on Malonyl-Coenzyme A Synthetase

Area of Science:

• Biochemistry
• Biotechnology
• Analytical Chemistry

Background:

• Adenosine triphosphate (ATP) is a crucial molecule in cellular energy transfer.
• Accurate and sensitive detection of ATP is vital for understanding metabolic processes.
• Existing ATP detection methods may require reagents or lack specificity.

Purpose of the Study:

• To develop a novel, reagentless fluorescent biosensor for ATP detection.
• To utilize a bacterial enzyme as a scaffold for biosensor construction.
• To achieve sensitive and selective ATP measurements.

Main Methods:

• Engineered malonyl-coenzyme A synthetase from Rhodopseudomonas palustris as the protein scaffold.
• Covalently attached two 5-iodoacetamidotetramethylrhodamine molecules to the protein.
• Measured changes in fluorescence intensity upon ATP binding.

Main Results:

• The biosensor exhibited a 3.7-fold increase in fluorescence intensity upon ATP binding.
• Achieved micromolar sensitivity for ATP detection.
• Demonstrated high selectivity for ATP over ADP.
• Successfully monitored enzymatic ATP production and depletion in kinetic assays.

Conclusions:

• A robust, reagentless fluorescent biosensor for ATP has been successfully developed.
• The biosensor provides a sensitive and selective method for ATP quantification.
• This tool is valuable for studying enzyme kinetics and cellular metabolism.