FRET Signal

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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!
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