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Fluorescence 1. 2. 3. 4. 5. 6. What is fluorescence Technical issues Uses of Fluorescence: Quantitation Ligand binding Conformational changes Measuring distances Fluorescence quenching Fluorescence lifetimes Fluorescence What happens after a molecule has adsorbed light exciting light photobleaching * excited state Lifetime 1 –10 10-9 secs (1-10 nsecs) (try to avoid) heat emission of fluorescence light Fluorescence excited state heat Energy heat ground state Absorption Stokes shift Intensity Abs Flu Wavelength (nm) Lowest vibrational level in excited state Fluorescence emission Fluorescence of Trp 280 nm 340 nm Intensity Abs Flu Wavelength (nm) Fluorescent amino acids: Trp, Phe, Tyr Fluorescence of proteins is dominated by Trp Fluorescence 1. 2. 3. 4. 5. 6. What is fluorescence Technical issues Uses of Fluorescence: Quantitation Ligand binding Conformational changes Measuring distances Fluorescence quenching Fluorescence lifetimes Fluorescence Instrumentation Spectrofluorimeter Looks at samples in solution (Typically 3 mls) High sensitivity ( conc 0.1 µM) Fluorescence microscope Looks at spatial distribution in 2D or 3D (confocal) Source of Radiation Arc lamp (450 watt Xenon) + monochromator Laser (generally fixed wavelength eg Argon-ion laser 488 nm Detector Phototube (can be electrically cooled) CCD camera on fluorescence microscopes Fluorimeter (right angle geometry) excitation monochromator light source slit cuvette unabsorbed exciting light slit fluorescence light emission monochromator fluorescence detector Sensitivity Down to ca 0.01 µM (ca 100 times more sensitive than absorption spectroscopy Limited by: Scattered light (problem with cloudy samples) Background fluorescence Impurities in buffers (use Ultra-pure reagents) Autofluorescence in cells Ideal fluorescence molecule: High extinction coefficient ε (Absorbs light well) High quantum yield Large Stoke’s Shift High wavelength of emission Usually contains multiple –C=C- Sensitivity Common problem : concentration too high Inner filter effect Flu. intensity Optical Density less than 0.05 to avoid inner filter effect cuvette Concentration Volume observed excitation Inner Filter Effect Absorption of light here leads to low light intensity in volume observed slit Flu light absorbed fluorescence light Fluorescence Parameters Stokes shift Intensity Abs Flu Wavelength (nm) Wavelength of maximum absorption (equiv. to abs. Spectrum) intensity of absorption – extinction coefficient ε Wavelength of maximum fluorescence emission environmentally sensitive Fluorescence quantum yield Q (related to intensity of emission) environmentally sensitive (cont.) Fluorescence Parameters (cont.) Fluorescence quantum yield Q (related to intensity of emission) Q= no. of photons emitted no. of photons absorbed Q between 0 and 1 (Trp Q ca 0.2) heat excited state heat ground state Fluorescence emission Absorption Fluorescence lifetime Time molecules remains in the excited state typically 1-10 nsecs Fluorescence Intensity Measurements Abs OD Flu Abs=εcl Fluorescence Intensity Arbitrary units Wavelength (nm) Fluorescence intensity is measured in ‘arbitrary units’ Size of signal depends on sensitivity of the fluorimeter Thus need to standardize: measure relative to some convenient standard solution Fluorimeter (right angle geometry) excitation monochromator light source slit cuvette unabsorbed exciting light slit fluorescence light emission monochromator fluorescence detector Fluorescence 1. 2. 3. 4. 5. 6. What is fluorescence Technical issues Uses of Fluorescence: Quantitation Ligand binding Conformational changes Measuring distances Fluorescence quenching Fluorescence lifetimes Uses of Fluorescence Quantitate material: Flu. Int. proportional to conc. Environmental change: Flu maximum can shift (spectral shift) Q can change (intensity change) (cont.) Intensity Abs Flu Wavelength (nm) Uses of Fluorescence (cont.) Conformational changes on a protein Trp fluorescence Labelled protein eg at a Cys residue with a fluorescence probe Ligand binding Changes in protein fluorescence Changes in ligand fluorescence if ligand is fluorescent Examples: Ca2+ binding to Ca2+ -ATPase Calcium ATPase Trp residues Ca2+ binding sites Ca2+-ATPase + Ca2+ Change in Trp fluorescence intensity 12 10 pH 8.5 ∆F/F (%) 8 6 4 2 0 2 3 4 5 6 pCa 7 8 9 10 Cys residues NBD-Cl Bromomethylcoumarin Cys-344 Lys-515 FITC NBD-labelled Ca2+-ATPase 0.7 mM Ca2+ 0.5 mM EGTA 5% 10 mM Mg2+ Uses of Fluorescence (cont.) Conformational changes on a protein Trp fluorescence Labelled protein eg at a Cys residue with a fluorescence probe Ligand binding Changes in protein fluorescence Changes in ligand fluorescence if ligand is fluorescent Examples: Ca2+ binding to Ca2+ -ATPase Location of Trp residues in a membrane protein Uses of fluorescence To report on environment Bacteriorhodopsin polar different λmax for Trp hydrophobic Trp fluorescence spectra 1.5 Intensity W Free Trp 1.0 0.5 0.0 300 320 340 360 Wavelength (nm) 380 400 Fluorescence 1. 2. 3. 4. 5. 6. What is fluorescence Technical issues Uses of Fluorescence: Quantitation Ligand binding Conformational changes Measuring distances Fluorescence quenching Fluorescence lifetimes Uses of Fluorescence – Distance Measurements Fluorescence Energy Transfer A r D Donor Int. Abs Flu Energy Transfer Acceptor Int. Flu Abs Wavelength (nm) Excite Flu emission (cont.) Uses of Fluorescence – Distance Measurements (cont.) Efficiency of Energy Transfer proportional to r6 A D Fluorescence emission spectra D alone Int. F0 r D+A F Efficiency of transfer E= 1- F/F0 Forster theory: E = R06/(r6 + Ro6) R0 = distance of separation at which E = 50% typically 50 Å Measure decrease in F for donor caused by the presence of the acceptor Energy Transfer IAEDANS to BrF 25 BrF E439 40 Å I n t e n s i ty 20 IAEDANS-BrF IAEDANS C670, C674 15 BrF IAEDANS 10 5 0 400 450 Wavelength (nm) 500 550 IAEDANS-ATPase to FITC-PE 1.0 C670, C674 F/Fo 0.8 h=54A EGTA 2+ Ca vanadate 0.6 Mg 0.4 2+ Thapsivillosin 0.2 0 0 0.1 0.2 0.3 Mole Fraction FITC-PE 0.4 Fluorescence Energy Transfer K-515 42 E-439 40 53 37 80 45 41 C-344 28 70 C-670/4 54 Uses of Fluorescence – Distance Measurements (cont.) Measure distances on a protein A r D Peptide Hydrolysis D A Molecular beacons (DNA or RNA) D D A A binding to DNA Uses of Fluorescence – Fluorescence quenching Short range – requires contact between fluorophore and quencher exciting light heat * emission of fluorescence light excited state exciting light * 100 % heat quencher Uses of Fluorescence – Fluorescence quenching Short range – requires contact between fluorophore and quencher Water soluble – iodide (I-), acrylamide, O2 Hydrophobic – aliphatic bromides Use to test accessibility Quench from Aqueous phase Quench from Lipid bilayer Trp containing peptide Dibromo-tyrosine containing peptide W DibromoQ Tyr 1.0 Fluorescence 0.8 no quenching C14 0.6 Thickness C18 0.4 C24 0.2 W Q 0.0 0 0.02 0.04 Mole fraction brominated peptide quenched Fluorescence 1. 2. 3. 4. 5. 6. What is fluorescence Technical issues Uses of Fluorescence: Quantitation Ligand binding Conformational changes Measuring distances Fluorescence quenching Fluorescence lifetimes Fluorescence lifetimes exciting light heat * excited state emission of fluorescence light Lifetime 1 –10 10-9 secs (1-10 nsecs) Measure using pulse methods Excite fluorescence with a short pulse of light Measure time between excitation and emission of a photon of light (cont) exciting light Fluorescence lifetimes (cont) * heat emission of fluorescence light No No. of photons emitted at time t Nt Exponential decay Nt=Noexp(-t/τ ) τ = fluorescence lifetime Time (t) (nsecs) Uses of Fluorescence lifetimes Environmentally sensitive protein changes etc. Protein change Often: Intensity Lifetime F1 F2 τ1 τ2 F1 F2 = τ1 τ2 Advantage of τ : this is an absolute measurement Disadvantage of F ; this is in arbitrary units, and varies with amount of protein