1. FRET(Fluorescent Resonance Energy Transfer)

2. The problem
The use of fluorescent probes (ex. GFP) permits direct observation of the dynamic properties of specific proteins in live cells .
But, It is difficult to observe functional information like protein-protein interaction using fluorescent probes.
when proteins are labeled with different fluorophores, the optical resolution of light microscopes limits the detection of protein proximities to about 0.2 µm.
when proteins are labeled with different fluorophores, the optical resolution of light microscopes limits the detection of protein proximities to about 0.2 µm

3. What is FRET?
FRET is a process that shifts energy from an electronically excited molecule(donor) to a neighboring molecule(acceptor).
If the two fluorophores are close enogh, then excitation of the first molecule (Donor) results in fluorescence emission of the second molecule (Acceptor).

4. What is FRET?
CFP is in close to YFP. (1-10 nm)

5. 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

6. transfer its excitation energy to a nearby acceptor chromophore in a non-radiative fashion through long-range dipole-dipole interactions.

7. Fluorophore pair for FRET
The donor emission spectrum must overlap significantly with the acceptor excitation spectrum.

8. 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
dsRed1
560nm
610nm
FITC
Cy3
525nm
595nm
Cy5
633nm
695nm
Rhodamine
543nm

9. A donor can directly transfer its excitation energy to an acceptor through long-range dipole-dipole intermolecular coupling.
A theory proposed by Theodor Förster in the late 1940s
Resonance energy transfer is a non-radiative quantum mechanical process (no collision, heat) When energy transfer occurs, the acceptor molecule quenches the donor molecule fluorescence, and if the acceptor is itself a fluorochrome, increased or sensitized fluorescence emission is observed

10. Rate constant , K Rate constant for energy transfer(Kt)
𝐾𝑡~ 𝑄𝐷𝐽 𝜏𝐷 × 1 𝑅6 ×𝜅2
𝐾𝑡 transfer rate
𝑄𝐷 quantum yield of donor
𝐽 spectral overlap of donor emission and acceptor absorption
𝜏𝐷 fluorescence lifetime of donor
R distance between donor and acceptor
𝜅2 orientational factor

11. FRET Efficiency Efficiency of Energy Transfer E = kT / (kT + kr + knr)
= 𝑑𝑒𝑎𝑐𝑡𝑖𝑣𝑎𝑡𝑖𝑜𝑛 𝑟𝑎𝑡𝑒 𝑏𝑦 𝑒𝑛𝑒𝑟𝑔𝑦 𝑡𝑟𝑎𝑛𝑠𝑓𝑒𝑟 𝑑𝑒𝑎𝑐𝑡𝑖𝑣𝑎𝑡𝑖𝑜𝑛 𝑟𝑎𝑡𝑒 𝑏𝑦 𝑎𝑙𝑙 𝑜𝑓 𝑡ℎ𝑒 𝑝𝑎𝑡ℎ𝑤𝑎𝑦𝑠
E = kT / (kT + kr + knr)
kT = rate of transfer of excitation energy
kr = rate of fluorescence(radiation)
knr = sum of the rates of all other deexcitation
processes (nonradiation)

12. R0 determination E = kT / (kT + kr + knr) Let, Then, E = 1 1+ 𝑅 𝑅0 6
R0 : distance between the donor and the acceptor at which 50% of FRET efficiency takes place.
Then, E = 𝑅 𝑅0 6
R0 (Forster distance) = 9.78 x 103(n-4*fd*k2*J)1/6 Å
depends on J(spectral overlap)

13. Structural design of various FRET-based biosensors.
The interaction of two proteins can be dynamically detected by FRET
Molecular cleavage by protease will be translated into loss of FRET.
An intramolecular probe consists of sandwiching two domains between CFP and YFP, which can interact after phosphorylation or binding to calcium, resulting in a change in FRET.
An intramolecular probe consists of CFP, YFP and a protein/domain, which permits conformational change by binding to another biomolecule, leading to a change in FRET

14. FRET quantification
FRET quantification is mostly based on measuring changes in fluorescence intensity or fluorescence lifetime upon changing the experimental conditions.
Ex>a microscope image of donor emission with normal acceptor and with acceptor bleached.

15. Various FRETs CFP-YFP pairs
-most popular FRET pair for biological use.
-CFP(cyan fluorescent protein)
-YFP(yellow fluorescent protein)
both are GFP variants.
They can be easily attached to a host protein by genetic engineering

16. Various FRETs BRET(Bioluminescence Resonance Energy Transfer)
-FRET require external illumination to initiate the fluorescence transfer.
-BRET use bioluminescent luciferase rather than CFP(donor) to produce an initial emission compatible with YFP.

17. Various FRETs Homo FRET
-to examine the interactions between two, or more proteins of the same type.
-both the acceptor and donor protein emit light with the same wavelenths.
-by FRET anisotropy imaging, measuring FRET is available.