A biosensor for fluorescent determination of ADP with high time resolution

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• ATP hydrolysis is fundamental to cellular processes, involving enzymes like kinases and ATPases.
• Direct measurement of enzymatic ATP conversion is limited, despite its biological significance.
• ADP is a common product released during ATP hydrolysis by various enzymes.

Purpose of the Study:

• To develop a novel, reagentless biosensor for the real-time detection of ADP.
• To create a sensitive probe for monitoring enzymatic ATP conversion.
• To overcome limitations in existing methods for measuring ATP hydrolysis products.

Main Methods:

• Engineered a biosensor using a bacterial actin homologue, ParM, as a protein framework.
• Attached a diethylaminocoumarin fluorophore to ParM to couple ADP binding with fluorescence.
• Mutated active site amino acids to reduce ATP affinity and enhance ADP specificity.
• Modified ParM to prevent filament formation and minimize intrinsic ATPase activity.

Main Results:

• Developed an ADP biosensor (MDCC-ParM) with high affinity (0.46 µM) and rapid response (0.65 µM⁻¹s⁻¹).
• Achieved >3.5-fold fluorescence increase upon ADP binding.
• Demonstrated >400-fold discrimination against ATP binding.
• Validated the biosensor's applicability in real-time kinetic assays for ATPases and a protein kinase.

Conclusions:

• The developed MDCC-ParM biosensor enables sensitive, real-time detection of enzymatically produced ADP.
• This reagentless probe offers a valuable tool for studying ATP-utilizing enzymes.
• The biosensor's specificity and sensitivity facilitate kinetic analysis of fundamental biochemical reactions.