Methods: Fluorescence Biochemistry 4000 Dr. Ute Kothe.
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Methods: Fluorescence Biochemistry 4000 Dr. Ute Kothe
Remember: Absorbance log (I 0 /I) = A = ε l c Beer-Lambert law Absorbance of monochromatic light reduces the intensity (I) Measured relatively to original intensity (I 0 ) 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 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 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 states due to spin conversion
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 Specific Solvent Effects: Chemical reactions of excited state with solvent, e.g. H-bonding, acid-base reactions etc. N: native state U: unfolded state
Quantifiying Binding Interactions Binding of Mant-GTP (●) and Mant-GDP (▲) to EF-G [Ligand] F = ------------------ [Ligand] + K D Environment of fluorophore, mant-nucleotide, changes upon binding to EF-G, i.e. the polarity of the surrounding changes and thus the fluorescence. K D 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 (R 0 ): Distance of 50% energy transfer Depends on dye pair Typically 30 – 60 Å Efficiency of energy transfer: R 0 6 E = -------------------- R 0 6 + r 6 r = distance between Donor and Acceptor