Methods: Fluorescence

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MethodsFluorescence

• Biochemistry 4000
• Dr. Ute Kothe

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Remember Absorbance

• Absorbance of monochromatic light reduces the
  intensity (I)
• Measured relatively to original intensity (I0)
• Depends on path length (l, often 1 cm),
  concentration (c) and molar extinction
  coefficient (e, units M-1 cm-1)
• Used to measure concentrations

Beer-Lambert law
log (I0/I) A e l c

• Is very fast provides information only on
  average ground state of molecules energy is set
  free by non-radiative decay (heat)

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Fluorophores
Often aromatic organic molecules Only atoms that
are fluorescent Lanthanides (europium, terbium)
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What is Fluorescence?
Upon excitation of a fluorophore, it re-emits
light at a longer wavelength.
Emission spectra typically independent of
excitation wavelength Excitation spectra
Wavenumber 1 / wavelength (linear to energy)
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Why Fluorescence?

• Highly sensitive -
• Detection in small quantities
• non-dangerous
• sensitive to environment
• Information on
• Interactions of solvent molecules with
  fluorophores
• Rotational diffusion of biomolecules
• Distances between sites on biomolecules
• Conformational changes
• Binding interactions
• Cellular Imaging
• Single-Molecule Detection

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Intrinsic Extrinsic Fluorophores

• Intrinsic Fluorophores
• Occur naturally
• Trp, Tyr, Phe
• NADH, FAD, FMN, Chlorophyll
• Etc.
• Extrinsic Fluorophores
• Added artifically to a sample
• Dyes binding DNA (ethidium bromide)
• Labelling of amino groups (dansyl
• chloride, fluorescein isothiocyanate)
• Labelling of sulfhydryl groups
• (maleimide dyes)
• etc.

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Fluorescence Spectrometer
Light Source Xenon Lamp or Laser Excitation
Monochromator Sample Cell Emission
Monochromator Detector Photomultiplier
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Fluorescence Plate Reader

• For fast high-throughput measurements in multiple
  well plates

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Jablonski Diagram
Allowed singlet states e- in excited orbital is
paried by opposite spin to second e- in
ground-state orbital
Forbidden triplet states due to spin conversion
Franck-Codon Principle all electronic
transitions occur without change in the position
of the nuclei (because they are too fast for
siginificant displacement of nuclei).
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Stokes Shift
The energy of emission is typically less than the
energy of absorption. Thus Fluorescence occurs at
longer wavelengths.
Fluorescence
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Solvent effects

• General Solvent Effects
• Fluorescence is highly dependend on solvent
  polarity!
• Tool to detect environement of fluorophor!
• Dipole moment of excited state larger than
  ground state
• solvent molecules reorient around excited dipole
• thus, solvent molecule lower the energy of the
  excited state
• Emission is shifted to longer wavelength

N native state U unfolded state
Specific Solvent Effects Chemical reactions of
excited state with solvent, e.g. H-bonding,
acid-base reactions etc.
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Quantifiying Binding Interactions
Binding of Mant-GTP (?) and Mant-GDP (?) to EF-G
Ligand F ------------------
Ligand KD

• KD determination

Environment of fluorophore, mant-nucleotide,
changes upon binding to EF-G, i.e. the polarity
of the surrounding changes and thus the
fluorescence.
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Resonance Energy Transfer
Transfer of energy from donor fluorophore to
acceptor molecule If donor emission spectra
overlaps with acceptor absorption spectra
No intermediate photon! D and A are coupled by
dipole-dipole interactions Distance Dependent
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Spectroscopic Ruler
Distance Dependence of Fluorescence Resonance
Energy Transfer (FRET) can be used to measure
distances between two dyes, e.g. Attached to
different interacting proteins.
Förster radius (R0) Distance of 50 energy
transfer Depends on dye pair Typically 30 60 Å
Efficiency of energy transfer R06 E
-------------------- R06 r6 r
distance between Donor and Acceptor
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Example Protein Interactions
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Nucleic Acid Detection

• Detection of nucleic acids by fluorescently
  labeled oligo-nucleotides
• Common dyes
• Cy3 Cy5
• Applications
• Molecular Beacons
• (see figure)
• cellular imaging
• microarrays
• etc.