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A biosensor for fluorescent determination of ADP with high time resolution.

ABSTRACT: Nearly every cellular process requires the presence of ATP. This is reflected in the vast number of enzymes like kinases or ATP hydrolases, both of which cleave the terminal phosphate from ATP, thereby releasing ADP. Despite the fact that ATP hydrolysis is one of the most fundamental reactions in biological systems, there are only a few methods available for direct measurements of enzymatic-driven ATP conversion. Here we describe the development of a reagentless biosensor for ADP, the common product of all ATPases and kinases, which allows the real-time detection of ADP, produced enzymatically. The biosensor is derived from a bacterial actin homologue, ParM, as protein framework. A single fluorophore (a diethylaminocoumarin), attached to ParM at the edge of the nucleotide binding site, couples ADP binding to a >3.5-fold increase in fluorescence intensity. The labeled ParM variant has high affinity for ADP (0.46 mum) and a fast signal response, controlled by the rate of ADP binding to the sensor (0.65 microm(-1)s(-1)). Amino acids in the active site were mutated to reduce ATP affinity and achieve a >400-fold discrimination against triphosphate binding. A further mutation ensured that the final sensor did not form filaments and, as a consequence, has extremely low ATPase activity. The broad applicability of N-[2-(1-maleimidyl)ethyl]-7-diethylaminocoumarin-3-carboxamide (MDCC)-ParM as a sensitive probe for ADP is demonstrated in real-time kinetic assays on two different ATPases and a protein kinase.

SUBMITTER: Kunzelmann S

PROVIDER: S-EPMC2785155 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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A biosensor for fluorescent determination of ADP with high time resolution.

Kunzelmann Simone S Webb Martin R MR

The Journal of biological chemistry 20090929 48

Nearly every cellular process requires the presence of ATP. This is reflected in the vast number of enzymes like kinases or ATP hydrolases, both of which cleave the terminal phosphate from ATP, thereby releasing ADP. Despite the fact that ATP hydrolysis is one of the most fundamental reactions in biological systems, there are only a few methods available for direct measurements of enzymatic-driven ATP conversion. Here we describe the development of a reagentless biosensor for ADP, the common pro ...[more]

PMID: 19801632

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Dataset Information

A biosensor for fluorescent determination of ADP with high time resolution.

Publications

A biosensor for fluorescent determination of ADP with high time resolution.

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