Fluorescent detection of GDP in real time with the reagentless biosensor, rhodamine-ParM

Simone Kunzelmann , Martin R Webb (1)

1 MRC National Institute for Medical Research

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, catalyzed especially by nucleoside triphosphatases and by kinases. Previously biosensors for ADP were developed in this laboratory, based on fluorophore adducts with variants of the bacterial actin homologue ParM. It is shown here 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.

Mots clés: Life Sciences

Simone Kunzelmann, Martin R Webb. Fluorescent detection of GDP in real time with the reagentless biosensor, rhodamine-ParM. Biochemical Journal, 2011, 440 (1), pp.43-49. ⟨10.1042/BJ20110349⟩. ⟨hal-00642832⟩