title: Fluorescence detection of GDP in real time with the reagentless biosensor rhodamine-ParM.
abstract: The development of novel fluorescence methods for the detection of key biomolecules is of great interest, both in basic research and in drug discovery. Particularly relevant and widespread molecules in cells are ADP and GDP, which are the products of a large number of cellular reactions, including reactions catalysed by nucleoside triphosphatases and kinases. Previously, biosensors for ADP were developed in this laboratory, based on fluorophore adducts with the bacterial actin homologue ParM. It is shown in the present study that one of these biosensors, tetramethylrhodamine-ParM, can also monitor GDP. The biosensor can be used to measure micromolar concentrations of GDP on the background of millimolar concentrations of GTP. The fluorescence response of the biosensor is fast, the response time being <0.2 s. Thus the biosensor allows real-time measurements of GTPase and GTP-dependent kinase reactions. Applications of the GDP biosensor are exemplified with two different GTPases, measuring the rates of GTP hydrolysis and nucleotide exchange.
authors.affiliation: MRC National Institute for Medical Research, Mill Hill, London NW7 1AA, UK.
authors.forename: Simone
authors.initials: S
authors.lastname: Kunzelmann
authors.forename: Martin R
authors.initials: MR
authors.lastname: Webb
mesh_terms: D000199:Actins / Q000378:metabolism; D015374:Biosensing Techniques / Q000379:methods*; D029968:Escherichia coli Proteins / Q000378:metabolism; D005456:Fluorescent Dyes; D020558:GTP Phosphohydrolases / Q000032:analysis; D006153:Guanosine Diphosphate / Q000032:analysis*; D012235:Rhodamines / Q000378:metabolism*; D018631:ras Proteins / Q000378:metabolism
chemical_list: D000199:Actins; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; C471255:ParM protein, E coli; D012235:Rhodamines; D006153:Guanosine Diphosphate; C005358:tetramethylrhodamine; D020558:GTP Phosphohydrolases; D018631:ras Proteins