FRET(Fluorescent Reso-nance Energy Transfer)

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 in-formation 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.

What is FRET?• FRET is a process that shifts energy

from an electronically excited mole-cule(donor) to a neighboring molecule(acceptor).

• If the two fluorophores are close enogh, then excitation of the first molecule (Donor) results in fluorescence emis-sion of the second molecule (Acceptor).

What is FRET?

CFP is in close to YFP. (1-10 nm)

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

• transfer its excitation energy to a nearby accep-tor chromophore in a non-radiative fashion through long-range dipole-dipole interactions.

Fluorophore pair for FRET• The donor emission spectrum must overlap signifi-

cantly 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 Ac-

ceptor

Emission Accep-

tor

CFP 440nm 480nm YFP 520nm 535nmBFP 365nm 460nm GFP 488nm 535nmCFP 440nm 480nm dsRed1 560nm 610nmFITC 488nm 535nm Cy3 525nm 595nmCy3 525nm 595nm Cy5 633nm 695nmGFP 488nm 535nm Rho-

damine543nm 595nm

• A donor can directly transfer its excitation energy to an acceptor through long-range dipole-dipole in-termolecular 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 mol-ecule quenches the donor molecule fluores-cence, and if the acceptor is itself a fluo-rochrome, increased or sensitized fluorescence emission is observed

Rate constant , K• Rate constant for energy transfer(Kt)

– transfer rate– quantum yield of donor– spectral overlap of donor emission and

acceptor absorption– fluorescence lifetime of donor– R distance between donor and acceptor– orientational factor

FRET Efficiency• Efficiency of Energy Transfer

=

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)

R0 determination• E = kT / (kT + kr + knr) • Let,

R0 : distance between the donor and the ac-ceptor at which 50% of FRET efficiency takes place.

• Then, E = R0 (Forster distance) = 9.78 x 103(n-4*fd*k2*J)1/6 Ådepends on J(spectral overlap)

Structural design of various FRET-

based biosen-sors.

a. The interaction of two proteins can be dynamically detected by FRET

b. Molecular cleavage by protease will be translated into loss of FRET.

c. An intramolecular probe consists of sandwiching two domains be-tween CFP and YFP, which can in-teract after phosphorylation or binding to calcium, resulting in a change in FRET.

d. 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

FRET quantification• FRET quantification is mostly based

on measuring changes in fluores-cence intensity or fluorescence life-time upon changing the experimental conditions.

Ex>a microscope image of donor emis-sion with normal acceptor and with accep-tor bleached.

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

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 com-patible with YFP.

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.