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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 associatedwith electronic transitionsAbsorbs one color light and emits another

Uses:Tracking molecules (i.e. proteins)

Give information about solute environmentMolecular ruler

Etc.

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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)

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Fluorescence Quantified

(Quantum Yield)

Number of photons fluoresced

Number of photons absorbed

f =

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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

1. Baseline reading of

fluorescing membrane

2. Photobleach to

destroy fluorescencein a spot

3. Monitor rates of

fluorescence recovery

4. Fluorescence recovery

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

http://www.me.rochester.edu/courses/ME201/webproj/FRAP.gifhttp://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

MolecularOxygen

Fluorophore

Molecular

Oxygen

Fluorescent Not

Fluorescent

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Fluorophore

Fluorescent

Iodide

High-energy vibration

states

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

D

ADEff

1

66

0

6

0

rrrEff

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

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

S0

T1

Intersystem crossing

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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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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-moleculeprotein-folding study. PNSA, 97(26), 14241-14244.

Olmsted, J, & Kearns, D. (1977). Mechanism of ethidium bromidefluorescence enhancement on binding to nucleicacids. Biochemistry, 16(16), 3647-3654.

Atherton, J, & Beaumont P. (1986). Quenching of the fluorescence ofDNA-intercalated ethidium bromide by some transition-metal ions. J. Phys.

Chem., 1986, 90(10), pp 22522259

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