Cfp

527 nm

Genetically engineered hybrid proteins

FIGURE 4 When the donor protein (CFP) is excited with monochromatic light of wavelength 433 nm, it emits fluorescent light at 476 nm (left). When the (red) protein fused with CFP interacts with the (purple) protein fused with YFP, that interaction brings CFP and YFP close enough to allow fluorescence resonance energy transfer (FRET) between them. Now, when CFP absorbs light of 433 nm, instead of fluorescing at 476 nm, it transfers energy directly to YFP, which then fluoresces at its characteristic emission wavelength, 527 nm. The ratio of light emission at 52 7 and 476 nm is therefore a measure of the interaction of the red and purple protein.

527 nm

Genetically engineered hybrid proteins

FIGURE 4 When the donor protein (CFP) is excited with monochromatic light of wavelength 433 nm, it emits fluorescent light at 476 nm (left). When the (red) protein fused with CFP interacts with the (purple) protein fused with YFP, that interaction brings CFP and YFP close enough to allow fluorescence resonance energy transfer (FRET) between them. Now, when CFP absorbs light of 433 nm, instead of fluorescing at 476 nm, it transfers energy directly to YFP, which then fluoresces at its characteristic emission wavelength, 527 nm. The ratio of light emission at 52 7 and 476 nm is therefore a measure of the interaction of the red and purple protein.

catalytic subunit (C) (Fig. 5). When these two hybrid proteins are expressed in a cell, BFP (donor; excitation at 380 nm, emission at 460 nm) and GFP (acceptor; excitation at 475 nm, emission at 545 nm) in the inactive PKA (R2C2 tetramer) are close enough to undergo FRET. Wherever in the cell [cAMP] increases, the R2C2 complex dissociates into R2 and 2C and the FRET signal is lost, because donor and acceptor are now too far apart for efficient FRET. Viewed in the fluorescence microscope, the region of higher [cAMP] has a minimal GFP signal and higher BFP signal. Measuring the ratio of emission at 460 nm and 545 nm gives a sensitive measure of the change in [cAMP]. By determining this ratio for all regions of the cell, the investigator can generate a false color image of the

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cAMP-dependent protein kinase (PKA)

380 nm cAMP-dependent protein kinase (PKA)

380 nm

cAMP S

(inactive) gfp 545 nm cAMP

cAMP S

(inactive) gfp 545 nm cAMP

380 nm

no emission at 545 nm

460 nm 433 nm

no emission at 545 nm

FIGURE 5 Measuring [cAMP] with FRET. Gene fusion creates hybrid proteins that exhibit FRET when the PKA regulatory and catalytic subunits are associated (low [cAMP]). When [cAMP] rises, the subunits dissociate, and FRET ceases. The ratio of emission at 460 nm (dissociated) and 545 nm (complexed) thus offers a sensitive measure of [cAMP].

Essentials of Human Physiology

Essentials of Human Physiology

This ebook provides an introductory explanation of the workings of the human body, with an effort to draw connections between the body systems and explain their interdependencies. A framework for the book is homeostasis and how the body maintains balance within each system. This is intended as a first introduction to physiology for a college-level course.

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