FRET and Other Energy Transfers

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FRET and Other Energy Transfers

• Patrick Bender

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Presentation Overview

• Concepts of Fluorescence
• FRAP
• Fluorescence Quenching
• FRET
• Phosphorescence

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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.

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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
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Fluorescence Quantified(Quantum Yield)

• Number of photons fluoresced
• Number of photons absorbed

Ff
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FRAP

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

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FRAP procedure

• Baseline reading of fluorescing membrane
• Photobleach to destroy fluorescence in a spot
• Monitor rates of fluorescence recovery
• Fluorescence recovery

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http//www.me.rochester.edu/courses/ME201/webproj/
FRAP.gif
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Fluorescence Quenching

• Environmental effect
• Solvent
• Additional solutes
• Other moieties
• Drastically effects quantum yield as well as rate
  of fluorescence

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How does it work?
Fluorophore
Fluorophore
Molecular Oxygen
Molecular Oxygen
Fluorescent
Not Fluorescent
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Fluorophore
Iodide
High-energy vibration states
Fluorescent
Radiationless energy transfer
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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

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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)

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FRET

• Forster Resonance Energy Transfer
• Involves radiationless energy transfer
• Used as molecular ruler
• Use in photosynthesis

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FRET

• Excitation of Donor
• Internal conversion of donor
• Excitation transfer of donor
• Fluorescence of acceptor

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What we can calculate

• Efficiency of transfer
• Distance between fluorophores (r)
• r0 Distance where efficiency equal 0.5

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http//www.olympusfluoview.com/applications/fretin
tro.html
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Photosystem II
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Phosphorescence

• Emission of light resulting from
  quantum-mechanically forbidden transitions
• Glow in the dark

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How it works
S1
Intersystem crossing
T1
S0
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Consequences

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

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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.
doi10.1016/j.ab.2009.10.048.
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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.
  doi10.1016/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/learni
  ng/applications/Fluorescence_Resonance/