Distance Dependence Of Fret

Fluorescence resonance energy transfer efficiency is inversely proportional to the sixth power of the distance between a donor and acceptor, and therefore is extremely

Fig. 1 A) General mechanisms of excited-state deactivation of a fluorophore. Up arrow represents transition from ground state (S0) to an Si excited state (thick line) or higher (thin lines) after photon absorption (wavy upward arrow). Down arrows represent relaxation, first from higher states down to Si (short arrow). Relaxation from S1 to S0 involves either fluorescence emission (F, wavy down arrow) or nonfluorescent mechanisms (NF, dashed down arrow). B) Mechanisms of excited state deactivation of a donor fluorophore in the presence of an acceptor. Same as A except that energy transfer (ET) is also displayed (longer dashed arrow) which results in less fluorescence emission from the donor excited state (D*) and activation of acceptor molecule from ground state (A) to excited state (A*). Excited state of A can also be depopulated via fluorescent (F) or nonfluorescent (NF) mechanisms.

Fig. 1 A) General mechanisms of excited-state deactivation of a fluorophore. Up arrow represents transition from ground state (S0) to an Si excited state (thick line) or higher (thin lines) after photon absorption (wavy upward arrow). Down arrows represent relaxation, first from higher states down to Si (short arrow). Relaxation from S1 to S0 involves either fluorescence emission (F, wavy down arrow) or nonfluorescent mechanisms (NF, dashed down arrow). B) Mechanisms of excited state deactivation of a donor fluorophore in the presence of an acceptor. Same as A except that energy transfer (ET) is also displayed (longer dashed arrow) which results in less fluorescence emission from the donor excited state (D*) and activation of acceptor molecule from ground state (A) to excited state (A*). Excited state of A can also be depopulated via fluorescent (F) or nonfluorescent (NF) mechanisms.

sensitive to their intermolecular separation. This is illustrated in the equation:

where E is FRET efficiency, r is the distance separating D from A, and R0 is the Forster distance. At the Forster distance, 50% of excited D is deactivated via FRET (ET in Fig. 1B), and the remainder is deactivated via fluorescence (F) and other nonfluorescent (NF) pathways. The Forster distance varies between different D-A pairs, typically ranging from 25 to 75 A (Table 1). R0 takes into consideration the amount of spectral overlap between the dyes and assumes that donors and acceptors are randomly oriented. For a donor-acceptor pair with an R0 of 50 A, doubling the intermolecular separation from 50 to 100 A results in a 32-fold decrease in FRET efficiency (Fig. 2B).

Fret Efficiency

Fig. 2 A) Fluorescence intensity plotted as a function of donor emission and acceptor absorption wavelengths, displaying a spectral overlap required for resonance energy transfer. B) FRET efficiency plotted as a function of the distance between a donor and acceptor pair characterized by a R0 of 50 A.

Fig. 2 A) Fluorescence intensity plotted as a function of donor emission and acceptor absorption wavelengths, displaying a spectral overlap required for resonance energy transfer. B) FRET efficiency plotted as a function of the distance between a donor and acceptor pair characterized by a R0 of 50 A.

Distance measurements obtained by monitoring FRET efficiency typically represent an average (rather than absolute) distance between D and A, because of the assumption of random orientation in the calculation of R0, and the fact that biological macromolecules usually fluctuate between thermodynamically equivalent states. Technicalities aside, the exquisite dependence of FRET on distances of the scale of biological macromolecules (Table 2) underscores the real usefulness of the technique: detecting biological phenomena involving changes in the

Table 1 Förster distances of commonly used FRET pairs

Donor

Acceptor

Ro (A)

Fluorescein

Tetramethylrhodamine

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