Methods:Fluorescence

Biochemistry 4000

Dr. Ute Kothe

Remember: Absorbance

log (I0/I) = A = ε l c

Beer-Lambert law

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

Is very fast & provides information only on average ground state

of molecules; energy is set free by non-radiative decay (heat)

Fluorophores

Often aromatic organic molecules

Only atoms that are fluorescent:Lanthanides (europium, terbium)

What is Fluorescence?

Emission spectra – typically independent of excitation wavelength

Excitation spectra

Upon excitation of a

fluorophore, it re-emits light

at a longer wavelength.

Wavenumber = 1 / wavelength (linear to energy)

Why Fluorescence?Highly sensitive -

Detection in small quantitiesnon-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

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.

Fluorescence Spectrometer

Light Source: Xenon Lamp or Laser

Excitation Monochromator

Sample Cell

Emission Monochromator

Detector: Photomultiplier

Fluorescence Plate Reader

For fast high-

throughput

measurements

in multiple

well plates

Jablonski Diagram

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).

Allowed singlet states:e- in excited orbital is paried by opposite spin to second e- in ground-state orbital

Forbidden triplet statesdue to spin conversion

Stokes ShiftThe energy of emission is typically less than the energy of absorption. Thus Fluorescence occurs at longer wavelengths.

Fluorescence

Solvent effectsGeneral 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

Specific Solvent Effects:Chemical reactions of excited state with solvent,e.g. H-bonding, acid-base reactions etc.

N: native stateU: unfolded state

Quantifiying Binding Interactions

Binding of Mant-GTP (●) and Mant-GDP (▲) to EF-G

[Ligand]F = ------------------ [Ligand] + KD

Environment of fluorophore, mant-nucleotide, changes upon binding to EF-G, i.e. the polarity of the surrounding changes and thus the fluorescence.

KD determination

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

Spectroscopic Ruler

Distance, Å

E

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 transferDepends on dye pairTypically 30 – 60 Å

Efficiency of energy transfer:R0

6

E = -------------------- R0

6 + r6 r = distance between Donor and Acceptor

Example: Protein Interactions

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.