1. Fluorescence Resonance Energy Transfer (FRET) Donor Fluorescence Acceptor Absorption INTENSITY 400450500550600650 WAVELENGTH (nm)

2. FRET (Fluorescence Resonance Energy Transfer) hv kFkF k NR S1S1 S0S0 kTkT k T = (1/  D )(r/R 0 ) 6 Förster Equation

3. Efficiency (E) of FRET E = k T /(k T + k D ) = R 0 6 /(R 0 6 + r 6 ) k D = relaxation rate in the absence of FRET = k F +  k NR = 1/  Donor or where R 0 = 8.79x10 -5 (  2 n -4  D J( 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. =  ε A ( λ )F D ( λ ) λ 4 d λ

4. Distance Dependence of FRET Efficiency = 1 – (I DA / I D ) INTENSITY 400450500550600650 WAVELENGTH (nm) A D R Efficiency 02468 Distance (nm) R 0 = 5.3 nm 0.5 1.0 10 Efficiency = R06R06 R 0 6 + R 6

5. Distance Dependence of FRET R 0 (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 – ( I DA / I D ) Or by looking at the sensitized emission of the acceptor molecule: E = (( I AD / I A ) – 1)(  A /  D )

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

8. HN Donor Acceptor

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

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

11. r R o 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) Efficiency 08 162432 R 0 =20 Å 0.5 1.0 40 R 0 6 I mant Nuct. R 0 6 + R 6 I Nuct. E = = 1– ATPADP The nucleotide binding pocket opens ~ 6Å upon phosphate release.

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