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FRET

FRET. 발표자 최예림. FRET 이란 무엇인가 ?. Fluorescence Resonance Energy Transfer 두 종류의 형광물질이 가까운 거리에 있을 때 공명에 의해 에너지를 전달하는 현상 10Å~100Å. 단백질의 크기나 세포막 두께 정도에 해당하는 거리. “ 거리 ＂ 의존적.

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FRET

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  1. FRET 발표자 최예림

  2. FRET이란 무엇인가? • Fluorescence Resonance Energy Transfer • 두 종류의 형광물질이 가까운 거리에 있을 때 공명에 의해 에너지를 전달하는 현상 • 10Å~100Å 단백질의 크기나 세포막 두께 정도에 해당하는 거리 • “거리＂의존적

  3. A donor can directly transfer its excitation energy to an acceptor through long-range dipole-dipole intermolecular coupling. • Proposed by Theodor Förster in the late 1940s • Energy transfer is non-radiative which means the donor is not emitting a photon which is absorbed by the acceptor

  4. FRET의 조건 • 형광물질 자체의 성질 • Donor와 acceptor 전이쌍극자의 배열 • Donor와 acceptor사이의 거리

  5. FRET의 조건-형광물질의 성질 The donor emission spectrum must overlap significantly with the acceptorexcitation spectrum

  6. Fluorophore pair for FRET • The excitation light for the donor must not directly excite the acceptor.

  7. FRET의조건-거리

  8. Förster Equation • Ro=foster critical distance = Distance at which energy transfer is 50% efficient = 9.78 x 103(n-4*fd*k2*J)1/6 Å = 2.11 × 10-2 • [κ2 • J(λ) • η-4 • QD]1/6 • fd : fluorescence quantum yield of the donor in the absence of acceptor • κ-squared : relative orientation in space between the transition dipoles of the donor and acceptor • J(λ) is the overlap integral in the region of the donor emission and acceptor absorbance spectra • η represents the refractive index of the medium • Q(D) is the quantum yield of the donor.

  9. J(λ) Fluorescnece Intensity Donor fluorescnece Acceptor absorption Wavelength

  10. a suitable scale for measurements in biological macromolecules and assemblies

  11. FörsterEquation Förster Equation

  12. Efficiency of Energy Transfer • E = 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

  13. FRET의 적용

  14. FRET의 장점 •FRET is relatively cheap •It is very efficient in measuring changes in distances. •Measure distances in molecules in solution. •Only need a few µM 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

  15. FRET의 단점 •The precision of the measure is impaired by the uncertainty of the orientation factor and by the size of the probes •When 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.

  16. Thank you!

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