FRET and Other Energy Transfers Patrick Bender. Presentation Overview Concepts of Fluorescence FRAP Fluorescence Quenching FRET Phosphorescence

  • View
    219

  • Download
    1

Embed Size (px)

Text of FRET and Other Energy Transfers Patrick Bender. Presentation Overview Concepts of Fluorescence FRAP...

  • Slide 1
  • FRET and Other Energy Transfers Patrick Bender
  • Slide 2
  • Presentation Overview Concepts of Fluorescence FRAP Fluorescence Quenching FRET Phosphorescence
  • Slide 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.
  • Slide 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)
  • Slide 5
  • Fluorescence Quantified (Quantum Yield) Number of photons fluoresced Number of photons absorbed f =
  • Slide 6
  • FRAP Fluorescence Recovery After Photo- bleaching Used to examine Brownian motion and 2-D interactions in membranes Examine molecular transport
  • Slide 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
  • Slide 8
  • http://www.me.rochester.edu/courses/ME201/webproj/FRAP.gif
  • Slide 9
  • Fluorescence Quenching Environmental effect Solvent Additional solutes Other moieties Drastically effects quantum yield as well as rate of fluorescence
  • Slide 10
  • How does it work? Fluorophore Molecular Oxygen Fluorophore Molecular Oxygen Fluorescent Not Fluorescent
  • Slide 11
  • Fluorophore Fluorescent Iodide High-energy vibration states Radiationless energy transfer
  • Slide 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
  • Slide 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)
  • Slide 14
  • FRET Forster Resonance Energy Transfer Involves radiationless energy transfer Used as molecular ruler Use in photosynthesis
  • Slide 15
  • FRET Excitation of Donor Internal conversion of donor Excitation transfer of donor Fluorescence of acceptor
  • Slide 16
  • What we can calculate Efficiency of transfer: Distance between fluorophores (r) r 0 = Distance where efficiency equal 0.5
  • Slide 17
  • http://www.olympusfluoview.com/applications/fretintro.html
  • Slide 18
  • Slide 19
  • Photosystem II
  • Slide 20
  • Phosphorescence Emission of light resulting from quantum- mechanically forbidden transitions Glow in the dark
  • Slide 21
  • How it works S1S1 S0S0 T1T1 Intersystem crossing
  • Slide 22
  • Consequences Violates quantum mechanics selection rules Inversion of spin Lifetime of excited triplet state in the millisecond or longer range
  • Slide 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.
  • Slide 24
  • Slide 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 22522259 Fluorescence resonance energy transfer (fret). (2010). Retrieved from http://www.andor.com/learning/applications/Fluorescence_Resonance/