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Some structures Dansyl chloride 1,5-I-AEDANS 5-(dimethylamino)naphthalene -1-sulfonyl chloride 5-[2-[(2-iodoacetyl)amino]ethylamino] naphthalene-1-sulfonic acid ANS Ethidium bromide Fluorescein isothiocyante Another family of probes The “fluorescent proteins” – Green fluorescent protein or GFP -S65-Y66-G67O2 T>30oC “fused” chromophore λex,max(nm) λem,max(nm) 398 508 H+ 398 nm 508 nm H+ Formation of the GFP fluorophore 2008 Nobel Prize in Chemistry Osamu Shimomura, Marine Biology Laboratory, Wood’s Hole Martin Chalfie, Biological Sciences, Columbia University Roger Tsien, HHMI, University of California, San Diego Let’s build a fluorimeter sample filter Light source slit detector Scanning excitation and emission spectra sample Light source monochromators Excitation (absorbance) and emission Spectra λmax,ex – excitation maximum λmax,em – emission maximum detector Scanning excitation and emission spectra I 260 320 Wavelength (nm) 380 440 Fluorescence intensity and concentration As the concentration increases the signal strength does not increase proportionately I expected Inner filter effect observed Aex + Aem Correction factor= antilog 2 Aex + Aem < 0.1 0.1 0.2 Concentration A Beware of changes to Aex + Aem (α A) Environment and fluorescence Emission λmax - exposure to more polar solvent will shift emission E.g., Tryptophan emission from protein, more polar environment longer λ emission Fluorescence Intensity Protein A λmax = 320 nm Protein B λmax = 350 nm Applications: Protein folding 300 350 Wavelength nm 400 Protein-protein, protein-ligand interactions Environment and fluorescence -2 Quantum yield decreases with exposure to more polar environment. e.g., ANS in a series of solvents Fluorescence Intensity octanol propanol methanol Application: ANS will partition into hydrophobic binding site on protein-this can be monitored by enhanced fluorescence ethylene glycol water 400 480 Wavelength nm 560 Fluorescence polarization III - I Polarization, P = III + I IIIand I -Intensity resolved parallel and perpendicular to excitation I -perpendicular to polraization of Incident light Light source monochromators Anisotropy, A = III - I III + 2 I III-parallel to the polarization of Incident radiation Depolarization and molecular motion III - I Anisotropy, A = III + 2 I Is measured as a fraction of the total fluorescence and is independent from the fluorophore concentration Anisotropy can be measured in steady-state and in time-resolved modes. Depolarization will occur as molecules rotate and this can be used to learn about molecular motion Protein + ligand Protein-ligand Rotational relaxation of protein ~ 10-100 ns Fluorescent, small molecule ligand ~ relaxation < 10 ns Time-averaged anisotropy of ligand will increase as it binds to the protein. Fluorescence quenching Dynamic and static quenching Dynamic quenching involves collisions with quencher molecules to depopulate the excited state. Static quenching involves complex formation between the quencher Recall, prior to excitation. and fluorophore ΦF = kF / (kF + ∑ki) = τ / τF Quantum yield in the presence of quencher Q (ΦF)Q = = kF / (kF + ∑ki+ k[Q]) Ratio of fluorescence intensities in the absence and presence of Q, ΦF/ (ΦF)Q = (kF + ∑ki+ k[Q]) / (kF + ∑ki) = 1 + (k[Q]/(kF + ∑ki)) = 1 k[Q]τ Dynamic quenching – Stern-Volmer constant Dynamic quenching usually measured as intensity in the absence and presence of quencher, Io/IQ = 1 + K[Q] Stern-Volmer equation K – Stern-Volmer constant e.g., O2 is a quencher of W Fluorescence (Io/IQ) 1.8 We can compare W in different proteins for their sensitivity to quenching by O2 1.4 1.0 0.04 0.08 0.12 [O2] (M) 0.16 Fluorescence resonance energy transfer (FRET) Energy transfer is a result of interaction between donor and acceptor molecules- does not involve emission of a photon. The extent of energy transfer depends on distance (and other factors) and has seen extensive use to assess donor/acceptor distance. Donor molecule absorbs a photon (i.e., excitation) but instead of fluorescing energy transfer occurs to a neighboring, acceptor molecule. The acceptor must have an acceptable enegertic match for it to undergo excitation (i.e., resonance)