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
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)
Ground state
Fluorescence Quantified (Quantum Yield)
Number of photons fluoresced f = Number of photons absorbed
FRAP
Fluorescence Recovery After Photobleaching 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
http://www.me.rochester.edu/courses/ME201/webproj/FRAP.gif
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 Iodide
Fluorescent
Radiationless energy transfer
High-energy vibration states
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:
D A Eff 1 D
Distance between fluorophores (r)
r0= Distance where efficiency equal 0.5
r06 Eff 6 6 r0 r
http://www.olympusfluoview.com/applications/fretintro.html
Photosystem II
Phosphorescence
Emission of light resulting from quantummechanically forbidden transitions Glow in the dark
How it works
S1
Intersystem crossing
T1
S0
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 cellpenetrating 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/
