Understanding Fluorescence and FRET in Biophysics: Rafts in Cell Membranes - Prof. Gail E., Study notes of Chemistry

Download Understanding Fluorescence and FRET in Biophysics: Rafts in Cell Membranes - Prof. Gail E. and more Study notes Chemistry in PDF only on Docsity! Big 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 Techniques 4 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 absorption Frank-Condon Principle and Leonard-Jones Potential First excited state 6 f Ground state 2 Total F ‘ar (a) Factors Governing Fluorescence Intensity 1) 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 quenchers 3) Intersystem Crossing to Triplet State. Quantum Yield : number of photons emitted/number of photons absorbed. Quenching hy—> — om Figure 15-1 Energy-level diagram of two chromophores; G and S, indicate the ground and first excited states, respectively (heavy lines). The vibrational levels are the thin lines. A. This molecule is capable of fluorescing by the transition (solid arrow) indicated in the diagram. After excitation, there are vibrational losses (wavy arrow} to the lowest level of the excited state and then emission from this state (dashed arrow). B. This molecule fails to fluoresce because the vibrational levels of G are higher than the lowest level of S; hence, there can be a nonradiative transition (horizontal wavy arrows) from §, to a vibrational level of G followed by nonradiative losses to the bottom of G (vertical wavy arrow). Table 8-3 Typical fluorescent probes Absorption Emission® Sensitivity Aux Emax Anax Tr Emax PF Probe* Uses (nm) x 10-3 (nm) or (nsec) x 107? Dansyl chloride Covalent attachment to 330 3.4 510 0.1 13 3.4 protein: Lys, Cys 1,5-I-AEDANS Covalent attachment to 360 6.8 480 0.5 15 34 protein: Lys, Cys Fluorescein Covalent attachment to 495 42 516 0.3 4 116 isothiocyanate (FITC) protein: Lys 8-Anilino-l-naphthalene = Noncovalent bindingto 374 6.8 454 0.98 16 67 sulfonate (ANS) proteins Pyrene, and various Polarization studies on 342 40 383 0.25 100 100 derivatives large systems Ethenoadenosine, and Analogs of nucleotides 300 2.6 410 0.40 26 10 various derivatives bind to proteins, incorporate into nucleic acids Ethidium bromide Noncovalent binding to 515 38 600 ~l 26.5 38 nucleic acids Proflavine Covalent attachment to 445 15 516 0.02 30 monosemicarbazide RNA 3’-ends * Values shown for $, and t, are near the maximum typically observed in biological samples at ambient temperature. Other (considerably smaller) values often are found. * Structures of these probes are shown in Figure 8-16. Sensitivity 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/rotations example: (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 lifetime Overlap Integral Cleavage re NO FRET Fic. 6. Enzyme assays based on FRET principle. Fluoragenic sub- strates are synthesized in which doncr and acceptor molecules are attached to monomers (e.g... amino acids, saccharides, nucleotides) and located within the FRET distance. Upen cleavage, the FRET efficiency drops to zero, the intensity of enor Inereases and the intensity of acceptor decreases, STOLLOSI ET AL hy (D) (A) Antigen Fic. 8. FRET-based homogeneous immunoassay for polyhaptenic anti- gen. Two monoclonal antibodies raised against the antigen and labeled respectively with donor and acceptor fluorophore. FRET is observed only in the presence of the antigen Fic. 7. FRET-based homogeneous, competitive immunoassay. Antibodies and antigens are labeled with donor and ac- ceptor molecules respectively, The ana- lyte is the unlabeled antigen in this competitive assay, The extent of quench- ing (FRET) decreases in the presence of the unlabeled antigen Fluorescence Anisotropy Plane 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 dynamics FRET with Anisotropy: A . . “= i — 4 ~? y Beh Po ©~ je ll —@2“@=L., “oe .