FLUORESCENT NUCLEOTIDES

My lab developed a series of fluorescent ATP and GTP analogues in a collaboration with John Corrie's group (NIMR).

These had various coumarins and varying linker lengths to their attachment on the ribose. Using 3'-aminoATP gave effectively a zero-length linker.

These gave fluorescence signals on binding to proteins such as myosins and helicases. In some cases, fluorescence changes occur during other steps in the ATPase cycle.

(Key ref: 75, 85, 104)

Some applications: (Ref: 77, 94, 121)

PHOSPHATE ASSAY

Unlike the Pi biosensors, this assay is based on absorbance and is a coupled-enzyme assay that has found wide application for steady-state assays of phosphate formation

The key component is MESG, a guanosine analogue, which on reaction with Pi gives an absorbance change at 340 nm.

The components (MESG and phosphorylase) are commercially available, but it is essential to use the correct type of phosphorylase, as described in the published work.

(Key ref: 42)

An application (Ref: 44)