Text of FRET and Other Energy Transfers Patrick Bender. Presentation Overview Concepts of Fluorescence FRAP...
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: Tracking molecules (i.e. proteins) Give information about solute environment Molecular ruler Etc.
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)
Fluorescence Quantified (Quantum Yield) Number of photons fluoresced Number of photons absorbed f =
FRAP Fluorescence Recovery After Photo- bleaching Used to examine Brownian motion and 2-D interactions in membranes Examine molecular transport
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
Fluorescence Quenching Environmental effect Solvent Additional solutes Other moieties Drastically effects quantum yield as well as rate of fluorescence
How does it work? Fluorophore Molecular Oxygen Fluorophore Molecular Oxygen Fluorescent Not Fluorescent
Fluorophore Fluorescent Iodide High-energy vibration states Radiationless energy transfer
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
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)
FRET Forster Resonance Energy Transfer Involves radiationless energy transfer Used as molecular ruler Use in photosynthesis
FRET Excitation of Donor Internal conversion of donor Excitation transfer of donor Fluorescence of acceptor
What we can calculate Efficiency of transfer: Distance between fluorophores (r) r 0 = Distance where efficiency equal 0.5
Phosphorescence Emission of light resulting from quantum- mechanically forbidden transitions Glow in the dark
How it works S1S1 S0S0 T1T1 Intersystem crossing
Consequences Violates quantum mechanics selection rules Inversion of spin Lifetime of excited triplet state in the millisecond or longer range
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.
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/