Fluorescence Resonance Energy Transfer (FRET)

Donor Fluorescence

Acceptor Absorption

INT

EN

SIT

Y

400 450 500 550 600 650WAVELENGTH (nm)

FRET(Fluorescence Resonance Energy Transfer)

hv kF kNR

S1

S0

kTkT = (1/D)(r/R0)

6 Förster Equation

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

HN

Donor

Acceptor

Title

F344W mant-ATP22.4 Å

F344W MDE

Dominguez et al. 1998

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.

0 20 40 60 80 100 120

rate

(se

c-1 )

0

50

100

150

200

250

300

350

ATPmant ATP

Max. rate = 150 sec-1

Slope = 3.3 sec-1 μM -1

Stopped-flow rates

[nucleotide] μM