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/