Chemical Biology: Stephen Miller Flourescence

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What is fluorescence?

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Occurs when a molecule relaxes to its ground state after exitation by emitting a photon

-Some organic compounds relax to the triplet state, then phosphoresce to return to ground, therefore not emitting a fluorescent photon.

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Quantum Yield

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What types of molecules flouresce?

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Conjugated pi-system (high e)
Rigid molecules that relax primarily through photon emission rather than non-radiatively

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Fluorescence brightness =

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e x F

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Some proterties of spectra

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E=hv

v=c/wavelength

Red=low E
Violet=High E

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Quenching

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Occurs when interactions with other molecules decrease flourescence (ie, another molecule absorbs emitted photon before it is seen)

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Photobleaching

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irreversible destruction of the fluorophore, often a result of reaction with singlet oxygen

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Types of quenching

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Collisional quenching: loss of excitation energy as heat rather than light

static quenching: formation of a stable complex with reduced flourescence (ie dimerization dyes)

Photoinduced electron transfer (PET)

Forster resonance energy transfer (FRET)

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Quenching by dimerization of dyes

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static quenching: requires van der Waals contact

typically alters absorption spectra and lowers quantum yield

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Quenching by PET

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photoinduced electron transfer

excitation of an electron into a higher energy state allows a donor to fill the lower energy state, and an acceptor at an intermediate energy state to accept the initial excited electron.

Short range (<5 A) - VDW contact

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Quenching by FRET

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Through-space; 10-100Ǻ, typically 20-70 Ǻ.

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common Fluorophor colors

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Blue, green, red

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First use of immunofluorescence

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In 1950 Albert H. Coons covalently attached fluorescein isothiocyanate (FITC) to an antibody

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Live cell imaging using fluorescence (not fluorescent proteins)

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Microinjection of fluorescently-labeled protein into cells allows live cell imaging.

Ex: X-Rhodamine-labeled tubulin allows imaging of microtubule dynamics

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GFP

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Green Fluorescent protein from Aequeria Victoria (crystal jelly) by Shimomura in the 1960s/1970s

First cloned by Prasher in 1992

first Expressed in E. coli: Chalfie, 1994


Can be expressed in cells

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Which AAs flouresce?

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tryptophan
tyrosine
phenylalanine

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Drawbacks of GFP

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takes time to fold
needs proper environment

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Mutating GFP

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scientists have made BFP, YFP, CFP

no RFP

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dsRED

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-Gln-Tyr-Gly chromophore
-Tetrameric, oligomerizes
-Intermediate green state
-Maturation takes > 10 hours!

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dsRED mutations

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QYG (DsRed, mRFP)
MYG (tomato)
CYG (tangerine, banana)
TYG (orange, strawberry, cherry)
MWG (honeydew)

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Near IR flours

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can be used in vivo (Image a whole mouse)

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Dynamic Protein Localization techniques using fluors

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FRAP
photocaged fluorophores
Kaede: a molecular highlighter

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FRAP

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Flourescence recovery after photobleaching

-used to study protein diffusion rates and compartmentalization

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photocaged fluorophores

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"cage" is released upon activation with a particular wavelength, releasing fluorophor

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Flourescent probes and sensors:

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Environmental sensitivity

Modulation of quenching (PET, FRET)

Latent fluorophores

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DNA binding dyes

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have large increase in fluorescence upon binding DNA (intercalation, minor groove binding, ect.)

EtBr, Hoechst, sybr green

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Solvatochromic dyes

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change colour according to the polarity of the liquid in which they are dissolved

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Quenching as a probe:

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Use reporter-quencher dual labeled probes

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PET quenching by electron-rich molecules:

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tryptophan and guanine

Must be in close proximity (VDW) <5 A

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PET-based sensors of zinc and NO

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2 common PET-based assays

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GTP binding using Bodipy-FL

Calcium sensors

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FRET uses

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There are nucleotide and peptide FRET sensors

sensitive enough to see intramolecular interactions and even protein activation states

FRET is commonly used in genetically encoded Ca sensors

CFP-YFP are a common FRET pair due to little wavelength overlap

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Latent Flourophores

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Commonly used Beta-lactam conjugated fluorophore, activated by Beta galactosidase cleavage