FRET 발표자 최예림 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 • 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 No FRET Signal •CFP 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 Signal •CFP 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 FRET의 조건 형광물질 자체의 성질 Donor와 acceptor 전이쌍극자의 배열 Donor와 acceptor사이의 거리 FRET의 조건-형광물질의 성질 The donor emission spectrum must overlap significantly with the acceptor excitation spectrum Fluorophore pair for FRET • The excitation light for the donor must not directly excite the acceptor. Donor Excitation Donor Emission Donor Acceptor Excitation Acceptor Emission Acceptor CFP 440nm 480nm YFP 520nm 535nm BFP 365nm 460nm GFP 488nm 535nm CFP 440nm 480nm dsRed1 560nm 610nm FITC 488nm 535nm Cy3 525nm 595nm Cy3 525nm 595nm Cy5 633nm 695nm GFP 488nm 535nm Rhodamine 543nm 595nm FRET의 조건-거리 • 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. Fluorescnece Intensity J(λ) Donor fluorescnece Acceptor absorption Wavelength a suitable scale for measurements in biological macromolecules and assemblies Förster Equation ~ F ~ d ~ 17 D 8 . 8 10 4 6 A 4 n R ~ D Förster Equation W DA kr 2 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 FRET의 적용 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 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. Thank you!