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 absorbedΦf =

FRAP

Fluorescence Recovery After Photo-bleaching

Used to examine Brownian motion and

2-D interactions in membranesExamine 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

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)

r0= Distance where efficiency equal 0.5

D

ADEff

1

660

60

rr

rEff

http://www.olympusfluoview.com/applications/fretintro.html

Photosystem II

Phosphorescence

Emission of light resulting from quantum-mechanically forbidden transitions

“Glow in the dark”

How it works

S1

S0

T1

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 2252–2259

Fluorescence resonance energy transfer (fret). (2010). Retrieved from http://www.andor.com/learning/applications/Fluorescence_Resonance/