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Fluorescence Resonance Energy Transfer (FRET)

Donor Fluorescence. Acceptor Absorption. INTENSITY. 400. 450. 500. 550. 600. 650. WAVELENGTH (nm). Fluorescence Resonance Energy Transfer (FRET). FRET ( F luorescence R esonance E nergy T ransfer). F örster Equation. S 1. k T = (1/  D )(r/R 0 ) 6. k F. k NR. k T. hv.

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Fluorescence Resonance Energy Transfer (FRET)

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  1. Donor Fluorescence Acceptor Absorption INTENSITY 400 450 500 550 600 650 WAVELENGTH (nm) Fluorescence Resonance Energy Transfer (FRET)

  2. FRET(Fluorescence Resonance Energy Transfer) Förster Equation S1 kT = (1/D)(r/R0)6 kF kNR kT hv S0

  3. Efficiency (E) of FRET E = kT/(kT + kD) = R06/(R06 + r6) kD = relaxation rate in the absence of FRET = kF + kNR = 1/Donor or where R0 = 8.79x10-5(2n-4DJ(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

  4. D A R 1.0 R0 = 5.3 nm Efficiency INTENSITY 0.5 0 2 4 6 8 10 Distance (nm) 400 450 500 550 600 650 WAVELENGTH (nm) Distance Dependence of FRET Efficiency = 1 – (IDA/ ID) R06 Efficiency = R06 + R6

  5. Distance Dependence of FRET R0 (nm)

  6. 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)

  7. Upper 50 kDa Subdomain W441F W625F F425W ELC Actin-Binding Cleft V413W ABL W29F W546M W36F W597F Lower 50 kDa Subdomain W512F

  8. H N Donor Acceptor

  9. Title F344W MDE F344Wmant-ATP 22.4 Å Dominguez et al. 1998

  10. ATP ADP F344W-MDE fluorescence emission spectra Normalized fluorescence Wavelength (nm) Wavelength (nm)

  11. R06 Imant Nuct. E = = 1– R06 +R6 INuct. 1.0 R0=20 Å Efficiency 0.5 0 8 16 24 32 40 Distance (Å) ATP ADP Analysis of FRET Data Ro Efficiency (%) r D (% apo) DA (% apo) ADP 81 ± 5 21.4 Å 30 ± 2 Å 6 ± 0.4 76 ± 4 26 ± 2.3 20.1 Å 24 ± 2 Å 76 ± 3 56 ± 4 ATP The nucleotide binding pocket opens ~ 6Å upon phosphate release.

  12. Stopped-flow rates Slope = 3.3 sec-1 μM -1 Max. rate = 150 sec-1 [nucleotide] μM

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