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FRET and Other Energy Transfers

FRET and Other Energy Transfers. Patrick Bender. Presentation Overview. Concepts of Fluorescence FRAP Fluorescence Quenching FRET Phosphorescence. Fluorescence. Basically the emission of light associated with electronic transitions Absorbs one color light and emits another Uses:

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FRET and Other Energy Transfers

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  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? • (Solid Arrow) Excitation from impinging photon • (Dotted Arrow) Internal conversion • (Dashed Arrow) Electronic relaxation and light emission Excited state • Note: • Emitted light has longer wavelength than impinging • Internal conversion really fast (picosecond vs. microsecond) Ground state

  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 • Baseline reading of fluorescing membrane • Photobleach to destroy fluorescence in a spot • Monitor rates of fluorescence recovery • 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 Fluorophore Molecular Oxygen Molecular Oxygen Fluorescent Not Fluorescent

  11. Fluorophore Iodide High-energy vibration states Fluorescent 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) r0= Distance where efficiency equal 0.5

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

  18. Photosystem II

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

  20. How it works S1 Intersystem crossing T1 S0

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

  22. 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.

  23. 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/

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