CAMP Measurements in Living Cells Using FRETBased Technology

A different strategy to detect cAMP in living cells exploits fluorescence resonance energy transfer (FRET). In this approach, FRET is used to measure the conformational change that occurs upon cAMP binding to cAMP-binding domains that have been appropriately engineered for this purpose. FRET is a quantum-mechanical event that occurs when two fluorophores are placed in close proximity to each other (<100 Á), provided that the emission spectrum of the fluorophore that acts as the "donor" overlaps the excitation spectrum of the "acceptor" fluorophore. Under these circumstances, part of the vibrational energy of the excited state of the "donor" is transferred to the "acceptor" that emits at its own wavelength. The efficiency of this process (E) depends on the distance (R) between donor and acceptor with an inverse sixth power law, as described by the Förster's equation: E=1/(1+(R/R0)6) (Förster 1948), where R0 is the distance at which half of the energy is transferred. According to the Förster's equation, the doubling of the distance between the two fluorophores, for example, from R0 to 2R0, decreases the efficiency of transfer from E=50% to E=1.5%. Therefore, FRET provides a very sensitive measure of intermolecular distance. If the two fluorophores are held together by proteins that undergo a conformational change upon binding to cAMP, FRET can be used to measure cAMP concentration.

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