Intrinsic Fluorescence of Tryptophan, GFP, and Quenching - Prof. Stefan Franzen, Study notes of Physical Chemistry

Download Intrinsic Fluorescence of Tryptophan, GFP, and Quenching - Prof. Stefan Franzen and more Study notes Physical Chemistry in PDF only on Docsity! 1 Fluorescence Intrinsic Fluorescence: Tryptophan Green Fluorescent Protein Quenching Mechanisms Fluorescent Energy Transfer Applications NC State University Mirror image relationship between absorption and fluorescence bands 0-0 0-10-2 0-3 0-4 1-0 2-0 3-0 4-0 Wavelength Energy Absorption Fluorescence Rhodamine is an example of a mirror image relationship Fluorescence lifetime and quantum yield The intrinsic lifetime for a single state is given by a single exponential with time constant t0: The quantum yield is the ratio of the molecules that decay by the fluorescent pathway to the total: N t = N 0 e– t/τ0 φ = k f k f + k other = ττ0 Caspases Caspases are cysteine proteases that are activated during apoptosis. As with other proteases they have an inactive form and an active form. Splicing and folding of caspases is required to initiate their function in controlled cell death. Shown in the figure is the structure of caspase-1. Note that the structure indicates that two subunits have been cleaved and are intermingled. This is the active form. Tryptophan: an intrinsic probe H2 N CH C CH2 OH O HN The absorption is a p-p* transition. 2 280 nm excitation F lu or es ce nc e 1 105 2 105 3 105 4 105 5 10 5 300 320 340 360 380 400 Wavelength (nm) 0 M urea 4 M urea 8 M urea A excitation 280 nm 0 5 10 4 1 10 5 1.5 10 5 2 10 5 2.5 10 5 300 320 340 360 380 400 Wavelength (nm) 4 M urea 0 M urea 8 M urea B excitation 295 nm F lu or es ce nc e Different spectra at 280 (aromatics) and 295 (tryptophan) Fluoresence of procaspase-3 (C163S) 295 nm excitation NC State UniversityDr. Clay Clark - Biochemistry Process ∆G (kcal/mol ) (-) m (kcal/mol/M) K1 3.1±0.5 1.37±0.19 K2 14.4±1.5 1.52±0.26 K3 12.5±0.4 2.32±0.2 Concentration dependence of folding curve is shown. Urea denatures the protein. There is clear evidence for two intermediates. Table 1. N2 I2 2I 2U K2K1 K3 0 0.2 0.4 0.6 0.8 1 0 1 2 3 4 5 6 7 8 Urea (M) 0.25 µM 2 µM 0.5 µM 1 µM R el at iv e Fl uo re sc en ce (3 20 n m ) Unfolding of procaspase-3 (C163S) NC State UniversityDr. Clay Clark - Biochemistry 1.6 2 2.4 2.8 3.2 3.6 4 4.4 10 100 1000 104 105 Time (seconds) Unfolded Protein Native ProteinA R el at iv e F lu or es ce nc e The protein is rapidly diluted and refolding is observed on three different exponential time scales. NC State University Refolding kinetics of procaspase-3 (C163S) Dr. Clay Clark - Biochemistry 0 0.2 0.4 0.6 0.8 1 0 1 2 3 4 5 6 7 8 Urea (M) Dimer Unfolded MonomerII2 0.25 µM 2 µM 2 µM 0.25 µM The thermodynamic intermediates are shown obtained from the equilibrium data. Kinetic schemes In a two state model there are no intermediates: kF kU U F In a this case the equilibrium constant is: U = unfolded F = folded K = F U = k FkU The time course for reaching equilibrium is: A t = A0e– k obst where kobs = kF + kU Kinetic schemes: one intermediate For one intermediate: k1 k-1 U I In a this case the equilibrium constants are: U = unfolded I = intermediate F = folded However, the time course for reaching equilibrium is biexponential. The kinetics depend on whether U, I or F is being observed. However, as a general rule the approach to equilibrium for N intermediates involves N+1 exponential rate constants. F k2 k-2 K = K 1K2 = I U F I = F U Then, several chemical transformations occur: the glycine forms a chemical bond with the serine, forming a new closed ring, which then spontaneously dehydrates. Finally, over the course of an hour or so, oxygen from the surrounding environment attacks a bond in the tyrosine, forming a new double bond and creating the fluorescent chromophore. Since GFP makes its own chromophore, it is perfect for genetic engineering. You don't have to worry about manipulating any strange chromophores; you simply engineer the cell with the genetic instructions for building the GFP protein, and GFP folds up by itself and starts to glow. GFP is formed by post- translational modification 5 Fluorophore 5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid, sodium salt (EDANS) ε-(4-dimethylaminophenylazobenzoyl) -L-lysine (DABCYL) Quencher Fluorescent resonant energy transfer (FRET) Fluorescence resonance energy transfer (FRET) is a distance-dependent interaction between the electronic excited states of two dye molecules in which excitation is transferred from a donor molecule to an acceptor molecule without emission of a photon. FRET is dependent on the inverse sixth power of the intermolecular separation, making it useful over distances comparable with the dimensions of biological macromolecules. Thus, FRET is an important technique for investigating a variety of biological phenomena that produce changes in molecular proximity. Energy transfer mechanism Donor Acceptor Fluorescein Rhodamine Donor Acceptor hν Fluorescein Rhodamine Energy transfer mechanism Donor Acceptor hν Fluorescein Rhodamine Energy transfer mechanism Primary Conditions for FRET • Donor and acceptor molecules must be in close proximity (typically 10–100 Å). • The absorption spectrum of the acceptor must overlap fluorescence emission spectrum of the donor. • Donor and acceptor transition dipole orientations must be approximately parallel. 6 Förster and Dexter mechanisms of energy transfer The Dexter mechanism involves overlap of wavefunctions and is essentially identical to the transition moment for absorption with the difference that the initial and final states involve two molecules. D*A → DA* The Forster mechanism is a dipole-dipole mechanism that can operate over long distances (up to 100 Å!). This is the mechanism that is commonly used in biology to determine the distance between two molecules. Stryer L, Haugland RP. PNAS 58, 719-726 (1967) The Förster Radius The rate constant for energy transfer is: kDA = (R0/R)6 The distance at which energy transfer is 50% is: Ro = [8.8 x 10-28κ2τ0-1n-4ΦJ(λ)]1/6 κ - orientation factor (2/3 for an isotropic sample) n - index of refraction Φ - quantum yield of the donor Spectral overlap integral is Donor Acceptor Ro (Å) Fluorescein Tetramethylrhodamine 55 IAEDANS Fluorescein 46 J λ = ε λ FD λ λ 4dλcm3M – 1 Wu P, Brand L. Anal Biochem 218, 1-13 (1994) Fluorescent resonant energy transfer probes of lipid mixing Membranes labeled with a combination of fluorescence energy transfer donor and acceptor lipid probes — typically NBD-PE and N-Rh-PE, respectively — are mixed with unlabeled membranes. Fluorescence resonance energy transfer, detected as rhodamine emission at ~585 nm resulting from NBD excitation at ~470 nm, decreases when the average spatial separation of the probes is increased upon fusion of labeled membranes with unlabeled membranes. The reverse detection scheme, in which fluorescence resonance energy transfer increases upon fusion of membranes separately labeled with donor and acceptor probes, has also proven to be a useful lipid mixing assay. Energy transfer strategy for the observation of membrane fusion Energy transfer (ET) is observed because D and A are close ET is not observed because D and A are too far apart Example of a donor/acceptor pair Nitrobenzoxadiazolyl (NBD) phosphatidyl ethanolamine Donor Rhodamine phosphatidyl ethanolamine Acceptor Excimer probes of membrane fusion Pyrene-labeled fatty acids can be biosynthetically incorporated into viruses and cells in sufficient quantities to produce the degree of labeling required for long-wavelength pyrene -excimer fluorescence. This excimer fluorescence is diminished upon fusion of labeled membranes with unlabeled membranes. Fusion can be monitored by following the increase in the ratio of monomer (~400 nm) to excimer (~470 nm) emission, with excitation at about 340 nm. 7 Excimer emission Excimer emission arises from the charge-transfer emission of two molecules in an excited state complex. 1) 2 mM pyrene (Argon) 2) 2 mM pyrene (Air) 3) 0.5 mM pyrene (Argon) 4) 2 µM pyrene (Argon) Py + Py + hνblue → Py + Py* → Py +:Py - → Py + Py + hνred Excimer emission as a probe of membrane fusion Excimer emission decreases after fusion