Fluorescence Resonance Energy Transfer (FRET)
Donor Fluorescence
Acceptor Absorption
INT
EN
SIT
Y
400 450 500 550 600 650WAVELENGTH (nm)
Efficiency (E) of FRET
E = kT/(kT + kD) = R06/(R0
6 + r6)
kD = relaxation rate in the absence of FRET = kF + kNR = 1/Donor
or
where R0 = 8.79x10-5(2n-4DJ(λ))1/6 = distance in Å at which E = 0.5.
n = refractive index 1.4 for protein solutions.2 = orientation factor = 2/3 for an isotropically tumbling system.D = quantum yield of donor.J (λ) = overlap integral between donor emission and acceptor
absorption. = εA(λ)•FD(λ)•λ4dλ
r RE
E
0
1 61
/
Distance Dependence of FRET
Efficiency = 1 – (IDA/ ID)
INT
EN
SIT
Y
400 450 500 550 600 650WAVELENGTH (nm)
ADR
Eff
icie
ncy
0 2 4 6 8Distance (nm)
R0 = 5.3 nm0.5
1.0
10
Efficiency = R0
6
R06 + R6
Distance Dependence of FRET
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 20 40 60 80 100
Distance (nm)
FR
ET
Eff
icie
ncy
20
40
60
R0 (nm)
Measurement of FRET
E can be experimentally measured by looking at changes in the emission lifetime or intensity (quantum yield) of the donor:
E = 1 – (DA/D)
= 1 – (IDA/ID)
Or by looking at the sensitized emission of the acceptor molecule:
E = ((IAD/IA) – 1)(A/D)
W29F
W36F
W512F
W546M
W597F
W441F W625F
V413W
ABL
ELC
Upper 50 kDa Subdomain
Actin-Binding Cleft
Lower 50 kDa Subdomain
F425W
Wavelength (nm)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
300 320 340 360 380 400
F344W
F344W + ATP
F344W + Mant ATP
Wavelength (nm)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
300 320 340 360 380 400
F344W
F344W + ADP
F344W + Mant ADP
F344W-MDE fluorescence emission spectra
ATP ADP
Nor
mal
ized
flu
ores
cenc
e
r Ro Efficiency (%) D (% apo) DA (% apo)
24 ± 2 Å 20.1 Å 26 ± 2.3 ADP 30 ± 2 Å 21.4 Å 6 ± 0.4
ATP
81 ± 5 76 ± 4 76 ± 3 56 ± 4
Analysis of FRET Data
Distance (Å)
Effi
ciency
0 8 16 24 32
R0=20 Å0.5
1.0
40
R06 Imant Nuct.
R06 + R6 INuct.
E = = 1–
ATP ADP
The nucleotide binding pocket opens ~ 6Å upon phosphate release.