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BOX 12-2 WORKING IN BIOCHEMISTRY (continued from previous page)

cell in which the ratio, or relative [cAMP], is represented by the intensity of the color. Images recorded at timed intervals reveal changes in [cAMP] over time.

A variation of this technology has been used to measure the activity of PKA in a living cell (Fig. 6). Researchers create a phosphorylation target for PKA by producing a hybrid protein containing four elements: YFP (acceptor); a short peptide with a Ser residue surrounded by the consensus sequence for PKA; a (P)-Ser-binding domain (called 14-3-3); and CFP (donor). When the Ser residue is not phosphor-ylated, 14-3-3 has no affinity for the Ser residue and the hybrid protein exists in an extended form, with the donor and acceptor too far apart to generate a FRET signal. Wherever PKA is active in the cell, it phosphorylates the Ser residue of the hybrid protein, and 14-3-3 binds to the (P>-Ser. In doing so, it draws YFP and CFP together and a FRET signal is detected with the fluorescence microscope, revealing the presence of active PKA.

FIGURE 6 Measuring the activity of PKA with FRET. An engineered protein links YFP and CFP via a peptide that contains a Ser residue surrounded by the consensus sequence for phosphorylation by PKA, and the 14-3-3 phosphoserine binding domain. Active PKA phos-phorylates the Ser residue, which docks with the 14-3-3 binding domain, bringing the fluorescence proteins close enough to allow FRET to occur, revealing the presence of active PKA.

FIGURE 6 Measuring the activity of PKA with FRET. An engineered protein links YFP and CFP via a peptide that contains a Ser residue surrounded by the consensus sequence for phosphorylation by PKA, and the 14-3-3 phosphoserine binding domain. Active PKA phos-phorylates the Ser residue, which docks with the 14-3-3 binding domain, bringing the fluorescence proteins close enough to allow FRET to occur, revealing the presence of active PKA.

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