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 μm) and a fast signal response, controlled by the rate of ADP binding to the sensor (0.65 μm−1s−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.
Highlights
An alternative approach is to take advantage of the highly specific interaction of the target molecule with a cognate binding protein
Screening Mutant/Fluorophore Combinations for a Fluorescence Response to ADP—Wild-type ParM contains two cysteine residues; Cys-100 is buried in a hydrophobic core and is probably inaccessible to labeling, whereas Cys-287 (Fig. 1) is solvent-exposed and likely to be modified by cysteine-reactive fluorophores
Having shown that the latter cysteine was not useful as a fluorophore site, it was mutated to alanine, and new cysteine residues were introduced by site-directed mutagenesis into this C287A variant
Summary
Plasmids—Plasmid pJSC1 for recombinant synthesis of wildtype ParM was provided by J. Titrations of MDCC-labeled ParM variants with adenine nucleotides were analyzed with a quadratic binding curve using Grafit software [18],. For calibration of the sensor response, MDCC-ParM at the concentration used in the assay was titrated with ADP, and the fluorescence was recorded. A gradient was obtained for each initial ATP concentration, corresponding to the change in fluorescence intensity per micromolar ADP. Instead of performing such a calibration for each initial ATP concentration used in the helicase or kinase assay, the titrations were performed at only four different total nucleotide concentrations. ADP binding kinetics of MDCC-ParM was measured in 30 mM Tris1⁄7HCl, pH 7.5, 25 mM KCl, 3 mM MgCl2, and 5 M bovine serum albumin. The rate constant was only 10% different between the measurements with 80 and 160 M MDCC-ParM
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