Methods:Fluorescence Biochemistry 4000 Dr. Ute Kothe
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 = ε l c 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? 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)
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
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 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).
Stokes Shift The energy of emission is typically less than the energy of absorption. Thus Fluorescence occurs at longer wavelengths. Fluorescence
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.
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.
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
E Distance, Å 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
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.