1. FRET and Other Energy Transfers Patrick Bender

2. Presentation Overview Concepts of Fluorescence FRAP Fluorescence Quenching FRET Phosphorescence

3. Fluorescence Basically the emission of light associated with electronic transitions  Absorbs one color light and emits another Uses:  Tracking molecules (i.e. proteins)  Give information about solute environment  Molecular ruler  Etc.

4. How does it work? Excited state Ground state 1.(Solid Arrow) Excitation from impinging photon 2.(Dotted Arrow) Internal conversion 3.(Dashed Arrow) Electronic relaxation and light emission Note: Emitted light has longer wavelength than impinging Internal conversion really fast (picosecond vs. microsecond)

5. Fluorescence Quantified (Quantum Yield) Number of photons fluoresced Number of photons absorbed Φf =

6. FRAP Fluorescence Recovery After Photo- bleaching Used to examine Brownian motion and 2-D interactions in membranes Examine molecular transport

7. FRAP procedure 1.Baseline reading of fluorescing membrane 2.Photobleach to destroy fluorescence in a spot 3.Monitor rates of fluorescence recovery 4.Fluorescence recovery

8. http://www.me.rochester.edu/courses/ME201/webproj/FRAP.gif

9. Fluorescence Quenching Environmental effect  Solvent  Additional solutes  Other moieties Drastically effects quantum yield as well as rate of fluorescence

10. How does it work? Fluorophore Molecular Oxygen Fluorophore Molecular Oxygen Fluorescent Not Fluorescent

11. Fluorophore Fluorescent Iodide High-energy vibration states Radiationless energy transfer

12. Examples of quenching Ethidium Bromide  Interchelated with DNA vs. in solvent  Interchelated with DNA in presence of other metals Fluorescence quenching by tryptophan  Locate fluorophore proximity to tryptophan

13. Quenchers Single molecule protein folding  Fluorescing molecules quench each other in folded conformation Common quenchers:  Water  Molecular Oxygen  Many electron molecules/ions (e.g. Iodide)

14. FRET Forster Resonance Energy Transfer Involves “radiationless” energy transfer Used as molecular ruler Use in photosynthesis

15. FRET Excitation of Donor Internal conversion of donor Excitation transfer of donor Fluorescence of acceptor

16. What we can calculate Efficiency of transfer: Distance between fluorophores (r) r 0 = Distance where efficiency equal 0.5

17. http://www.olympusfluoview.com/applications/fretintro.html

19. Photosystem II

20. Phosphorescence Emission of light resulting from quantum- mechanically forbidden transitions “Glow in the dark”

21. How it works S1S1 S0S0 T1T1 Intersystem crossing

22. Consequences Violates quantum mechanics selection rules  Inversion of spin Lifetime of excited triplet state in the millisecond or longer range

23. Uses Can be used to test for presence of oxygen species in different environments  Non-invasive  Examine mitochondrial function and energy levels of cells Dmitriev, R., Zhdanov, A., Ponomarev, G., Yashunski, D., & Papkovsky, D. (2010). Intracellular oxygen-sensitive phosphorescent probes based on cell-penetrating peptides. Analytical Biochemistry, 398(1), 24-33. doi:10.1016/j.ab.2009.10.048.

25. List of Works Cited Dmitriev, R., Zhdanov, A., Ponomarev, G., Yashunski, D., & Papkovsky, D. (2010). Intracellular oxygen-sensitive phosphorescent probes based on cell- penetrating peptides. Analytical Biochemistry, 398(1), 24-33. doi:10.1016/j.ab.2009.10.048. Zhuang, X. et al. (2000). Fluorescence quenching: a tool for single-molecule protein-folding study. PNSA, 97(26), 14241-14244. Olmsted, J, & Kearns, D. (1977). Mechanism of ethidium bromide fluorescence enhancement on binding to nucleic acids. Biochemistry, 16(16), 3647-3654. Atherton, J, & Beaumont P. (1986). Quenching of the fluorescence of DNA-intercalated ethidium bromide by some transition-metal ions. J. Phys. Chem., 1986, 90 (10), pp 2252–2259 Fluorescence resonance energy transfer (fret). (2010). Retrieved from http://www.andor.com/learning/applications/Fluorescence_Resonance/