Format

Send to

Choose Destination
Biochemistry. 2006 Dec 12;45(49):14764-71.

A biosensor for inorganic phosphate using a rhodamine-labeled phosphate binding protein.

Author information

1
MRC National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom.

Abstract

A novel biosensor for inorganic phosphate (Pi) has been developed based on the phosphate binding protein of Escherichia coli. Two cysteine mutations were introduced and labeled with 6-iodoacetamidotetramethylrhodamine. When physically close to each other and correctly oriented, two rhodamine dyes interact to form a noncovalent dimer. In this state, they have little or no fluorescence, unlike the high fluorescence intensity of monomeric rhodamine. The labeling sites were so placed that the distance and orientation between the rhodamines change as a consequence of the conformational change associated with Pi binding. This movement alters the extent of interaction between the dyes. The best mutant of those tested (A17C, A197C) gives rise on average to approximately 18-fold increase in fluorescence intensity as Pi binds. The kinetics of interaction with Pi were measured at 10 degrees C. Under these conditions, the fluorescence increase associated with Pi binding has a maximum rate of 267 s-1. The Pi dissociation rate is 6.6 s-1, and the dissociation constant is 70 nM. An application of the sensor is demonstrated for measuring ATP hydrolysis in real time as a helicase moves along DNA. Advantages of the new sensor are discussed, both in terms of the use of a rhodamine fluorophore and the potential of this double labeling strategy.

PMID:
17144669
DOI:
10.1021/bi060960j
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for American Chemical Society
Loading ...
Support Center