FRET

FRET ?Fluorescence Resonance Energy Transfer 10~100

A donor can directly transfer its excitation energy to an acceptor through long-range dipole-dipole intermolecular coupling.

Proposed by Theodor Frster in the late 1940sEnergy transfer is non-radiative which means the donor is not emitting a photon which is absorbed by the acceptor

No FRET SignalCFP is excited by light and emits light CFP is more than 10 nm distant from YFP YFP is not excited and does not emit light

FRET SignalCFP is excited by light but does emit little light CFP is in close proximity (1-10 nm) to YFP YFP is not excited by light but does emit light

FRET Donor acceptor Donor acceptor

FRET - The donor emission spectrum must overlap significantly with the acceptor excitation spectrum

Fluorophore pair for FRETThe excitation light for the donor must not directly excite the acceptor.

DonorExcitation DonorEmission DonorAcceptorExcitation AcceptorEmission AcceptorCFP440nm480nmYFP520nm535nmBFP365nm460nmGFP488nm535nmCFP440nm480nmdsRed1560nm610nmFITC488nm535nmCy3525nm595nmCy3525nm595nmCy5633nm695nmGFP488nm535nmRhodamine543nm595nm

FRET -

Frster EquationRo=foster critical distance = Distance at which energy transfer is 50% efficient = 9.78 x 103(n-4*fd*k2*J)1/6 = 2.11 10-2 [2 J() -4 QD]1/6 fd : fluorescence quantum yield of the donor in the absence of acceptor -squared : relative orientation in space between the transition dipoles of the donor and acceptor J() is the overlap integral in the region of the donor emission and acceptor absorbance spectra represents the refractive index of the mediumQ(D) is the quantum yield of the donor.

a suitable scale for measurements inbiological macromolecules and assemblies

Frster Equation

Efficiency of Energy TransferE = kT/(kT + kf + k) kT = rate of transfer of excitation energy kf = rate of fluorescence k = sum of the rates of all other deexcitation processes (nonradiation)E = R60/ R60+ R6

FRET

FRET FRET is relatively cheapIt is very efficient in measuring changes in distances.Measure distances in molecules in solution.Only need a few M of labeled proteins.Once you have labeled your molecule, you can have a measurement rapidly.You can measure distances or changes in distances in a complex of molecules

FRET The precision of the measure is impaired by the uncertainty of the orientation factor and by the size of the probesWhen measuring a change in distance between two probes, the result is a scalar and give no indications of which probe (donor and/or acceptor) moves.These measurements give the average distance between the two probes.

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