Slide 1Slide 2Slide 3Slide 4Slide 5Slide 6QuenchingSlide 8Slide 9Slide 10Slide 11FRET: Fluorescence Resonance Energy TransferSlide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Big Question: We can see rafts in Model Membranes (GUVs or Supported Lipid Bilayers, LM), but how to study in cells? Do rafts really exist in cells? Are they static large structures? Are they small transient structures?FRET and FRET based Microscopy Techniques4 basic rules of fluorescence for overview presentation:•The Frank-Condon Principle: the nuclei are stationary during the electronic transitions, and so excitation occurs to vibrationally excited electronic states.•Emission occurs from the lowest vibrational level of the lowest excited singlet state because relaxation from the excited vibrational energy levels is faster than emission•The Stokes Shift: emission is always of lower energy than absorption due to nuclear relaxation in the excited state•The mirror image rule: emission spectra are mirror images of the lowest energy absorptionStokes shift is the difference (in wavelength or frequency units) between positions of the band maxima of the absorption and luminescence spectra of the same electronic transition.When a molecule or atom absorbs light, it enters an excited electronic state. The Stokes shift occurs because the molecule loses a small amount of the absorbed energy before re-releasing the rest of the energy as luminescence. This energy is often lost as thermal energy.Jablonski DiagramFluorescenceE = hhcFrank-Condon Principle and Leonard-Jones PotentialFactors Governing Fluorescence Intensity1) Internal conversion – non radiative loss via collisions with solvent or dissipation through internal vibrations. In general, this mechanism is dependent upon temperature. As T increases, the rate of internal conversion increases and as a result fluorescence intensity will decrease.2) Quenching – interaction with solute molecules capable of quenching excited state. (can be various mechanisms)O2 and I- are examples of effective quenchers3) Intersystem Crossing to Triplet State.Quantum Yield : number of photons emitted/number of photons absorbed.QuenchingCommon Fluorescence Applications in Biophysics:Tryptophan Fluorescence – Protein Folding/Binding IsothermsFluorescence Quenching - Protein Structure and DynamicsFluorescence Anisotropy – BindingFluorescence Resonance Energy Transfer – Binding, Distances, ConformationsCommon Fluorescent ProbesSensitivity to Local Environment:Fluorescence can be used to probe local environment because of the relatively long lived singlet excited state.10-9 to 10-8 sec, various molecular processes can occur•Protonation/deprotonation•Solvent cage reorganization•Local conformational changes•Translations/rotationsexample: (a) intensity and wavelength of fluorescence can change upon going from an aqueous to non-polar environment. This is useful for monitoring conformational changes or membrane binding.(b) Accesibility of quenchers, location on surface, interior, bilayer etc.FRET: Fluorescence Resonance Energy Transfer•Sensitive to interactions from 10 to 100Å•Increase acceptor sensitivity•Quenches donor fluorescence•Decreases donor lifetimeOverlap IntegralTransition DipolesFRET: Fluorescence Resonance Energy TransferRate:kt=d-1(Ro/R)6kt =rate of rxnd =lifetime of donorR=distance between fluorophoresRo =Förster distanceFörster distanceRo=(2*J()*n-4*Q)1/6*9.7*102J() =overlap integral2 =transition dipoles of fluorophoresn=refractive index of mediumQ=quantum yield% transfer = Efficiency (E):Quenching: E= 1-(I/Io)Energy Transfer: E=(ad(1)/da(1))*[(Iad(2)/Ia(2))-1]I=intensity with FRETIo=intenstiy without FRETad=absorption of accepter (with donor)da=absorption of donor (with acceptor)Fluorescence AnisotropyPlane polarized light to exite, detect linearly polarized light. Any motion that occurs on the time scale of the lifetime of the excited state, can modulate the polarization. Hence, this technique is used to measure size, shape, binding and conformational dynamicsFRET with Anisotropy:Fret Apps to BilayersGM1 toxinGPI anchored proteinsGFPHomo versus Hetero FretFRET fluorescence resonance energy
View Full Document
Unlocking...