A biosensor for fluorescent determination of ADP with high time resolution.
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.
MRC National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom.
Simone
S
Kunzelmann
Martin R
MR
Webb
D000199:Actins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001667:Binding, Competitive; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D003374:Coumarins / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence / Q000379:methods*
D000199:Actins; D003374:Coumarins; D029968:Escherichia coli Proteins; C471255:ParM protein, E coli; C088410:N-(2-(1-maleimidyl)ethyl)-7-(diethylamino)coumarin-3-carboxamide; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D000251:Adenosine Triphosphatases