1. Measurement of Fluorescent Lifetimes: Time-Domain Time-Correlated Single Photon Counting (TCSPC) vs. Stroboscopic (Boxcar) Techniques.

2. Deconvolution F(t) =  i e -t/  I S(t) =  L(t)F(t)dt

3. Time-Resolved Emission Spectra

4. Measurement of Fluorescent Lifetimes: Frequency-Domain

5. Exciting Light: L(t) = a + bsin(  t) (  = 2  x freq.) Emitted Light: F(t) = A + Bsin(  t -  ) Lifetimes: tan  =  x  p m = (B/A)/(b/a) = [1 +  ²  m ²] -½

6. Measurement of Fluorescent Lifetimes: Frequency-Domain

7. HN Fluorescent Molecules

8. HN Intrinsic Fluorescent Probes (i.e. tryptophan): Sensitive to local environment Relatively small Readily available in proteins Generated by site-directed mutagenesis Covalent Extrinsic Probes (i.e. TAMRA): Broad range of spectral properties Bright, relatively photostable Well characterized conjugation chemistry Non-covalent Probes (i.e. mant-ATP): Similar properties to covalent probes No need to permanently modify protein Target active site or ligand binding sites Fluorescent Molecules

9. Intrinsic Fluorophores

11. Extrinsic Fluorophores

12. Protein Structural Dynamics: effects on fluorescence emission spectra Polarization experiments are sensitive to changes in orientation of a fluorescent probe. Spectral Shifts depend on the environment around a fluorescent probe. A more polar environment tends to red shift the emission spectrum and a less polar environment tends to blue shift the emission spectrum. Dynamic Quenching experiments are a quantitative way to measure the accessibility of a fluorescent probe to quenching molecules in the solvent. FRET (Fluorescence Resonance Energy Transfer) experiments can measure the distance between a donor probe and an acceptor probe on the protein.

13. Dynamic Quenching: measure accessibility to solvent and rates of diffusion. k q - bimolecular quenching constant, proportional to rate of diffusion of quencher or fluorophore.  = k F /(k F +  k NR )  Q = k F /(k F +  k NR + k q [Q]) k q [Q] = pseudo-first order rate constant (M -1 s -1 ) since [Q] >> [F].  /  Q = (k F +  k NR + k q [Q])/ (k F +  k NR ) = 1 + k q [Q]/ (k F +  k NR ) and since  = 1/(k F +  k NR )  /  Q = 1 + k q [Q]  Stern-Volmer Equation: F 0 /F = 1 + K D [Q] K D = k q  = Stern-Volmer quenching constant. Plot of (F 0 /F - 1) vs. [Q] is linear with slope = K D. hv kFkF k NR k q [Q] S1S1 S0S0

14. F 0 /F = = 1 + k q [Q]  = 1 + K D [Q] Stern-Volmer Plots

15. Static Quenching F 0 /F = 1 + K S [Q] K s = equilibrium constant for quencher binding to fluorophore ([F-Q]/[F][Q]). Static quenching can be differentiated from dynamic quenching by: 1.) lifetime measurements - static quenching alters intensity, not lifetime. dynamic quenching alters both. 2.) temperature effects –

16. Combined Dynamic and Static Quenching: Stern-Volmer plot is concave upward. F 0 /F = (1 + K D [Q]) x (1 + K S [Q]) = 1 + (K D + K S )[Q] + K D K S [Q] 2 = 1 + K app [Q] therefore

17. Combined Dynamic and Static Quenching MAC

18. Therefore Quenching Sphere of Action

19. Two Populations of Fluorophores: one accessible to solvent, one not. F = F a + F b = (F 0a /(1 + K a [Q])) + F 0b (F 0 /(F 0 – F)) = 1/(ƒK[Q]) +1/ƒ where ƒ = F 0a /( F 0a + F 0a ), a = accessible and b = buried.

20. Electrostatic Effects on Dynamic Quenching

21. Dynamic Quenching – Two Populations of Fluorophores

22. 1. Apoazurin Pf1 2. Ribnuclease T 1 3. Staphylococcus nuclease 4. Glucagon

23. Collisional Quenching in Proteins Buried Residue Exposed Residue