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Fluorescence Spectroscopy Part II. Quenching Techniques Quenching • Quenching any process which reduces the lifetime of the excited state. reduction in the lifetime usually implies a decrease in the quantum yield Quencher • Collisional and static quenching require contact between the fluorophore and the quenching. Thus, these methods are useful to measure rates of diffusion and exposure of fluorescent species to the quencher. • A large number of quenchers are known and a partial list is: molecular oxygen, amides, BrO4-, xenon, I-, peroxides, nitroxides, acrylamide Quencher In protein study • Iodide. its charged nature quenches surface residues efficiently. • Oxygen. Small and can penetrate the protein to some degree. • Acrylamide, nitroxides. neutral. Mechanism of Quenching Collisional or dynamic quenching Static quenching Quenching by energy transfer Charge transfer reactions Dynamic Quenching When quenching occurs by a collisional mechanism, the quenching is an additional process that deactivates the excited state besides radiative emission. Stern-Volmer Relation FF fluorescent intensity in the absence of quencher FFQ fluorescent intensity in the presence of quencher t fluorophore decay lifetime in the absence of Q kq the quenching constant Let KSV = kqt Stern-Volmer Equation F0 1 K SV quencher F Stern-Volmer Relation F0 1 K SV quencher F •Since KSV = kqt if t is known, then kq values can be obtained from the slope of a plot of F0/F vs. [quencher]. •If quenching results from a nonfluorescent ground state complex between the fluorophore and the quencher, the dependence of F0/F on [quencher] is identical to that observed for dynamic quenching except the quenching constant is now the association constant. Stern-Volmer Plot Application of quenching Techniques •study of proteins is to determine the location of Trp residues •change in location of Trp residues, due to conformational changes. Reduction in the quenching of Trp residues bound to proteins versus that of free Trp is observed, and this can arise from two factors. •quenching of Trp is reduced to that of free Trp (or indole) because the Trp is now attached to a molecule with a smaller diffusion co-efficient. For proteins greater than 50 KDa in size, this results in a decrease in the quenching constant of 50. •Trp residue is buried in the protein and is thus not accessible to quenching agent unless the quenching agent can diffuse into the protein interior. •The correlation between acrylamide quenching and the emission wavelength of the Trp residue suggests that Trp residues buried in hydrophobic regions of the protein are less accessible for quenching. •The accessible fluorophores experience a decrease in fluorescence upon collision with collisional quenchers. •Charge versus steric effect is differentiated by quenchers with different size and charges. •Accessibility depends on exposure as well as lifetime. •Accessdibility of residues are reflected in the Stern-Volmer constant •For single tryptophan containing proteins, low values of kq indicate residues of low exposure, ie those buried within the protein structure. •These residues also have blue shifted emissions indicating a relatively nonpolar environment. F F* hexc F* kr F *+Q F* 0 kQ[Q] kIC kr F + hem Fluorescence F + Q Quenching F + heat Internal conversion k r k IC kQ [Q ] kr k r k IC without quencher kQ 0 I 0 t0 1 [Q] 1 t0 kQ [Q] kr k IC I t Example 1 Apoazurin Ade has two Trp residues, one surface residue and one buried residue. In the presence of iodide (collisional quencher), the spectrum resembles the structured emission seen in non-polar environments. The spectrum of the quenched Trp can be seen from the difference spectrum (dashed line), and is characteristic of an exposed residue in a partially hydrophobic environment. Example 2 Accessibility of Trp Residues in Lysozyme contains six tryptophan residues. positions 28, 62, 63, 108, 111 and 123. portion of these residues exposed to the aqueous solvent can be determined by Stern-Volmer equation . Fo = Fo,a + Fo,b F = F o,a / (1+KSV[Q]) + F o,b dF = Fo - F = Fo,aKSV[Q] / (1+KSV[Q]) Fo / (F0-F) = 1 / (KSV [Q]) + 1/fa fa is the fraction of accessible fluorophores obtained from intercept KSV can be obtained form the slope Example 3 Trp in Chymosin Example 3 Trp in Chymosin Example 4 Interaction of Tritrpticin with micelle Example 4 Interaction of Tritrpticin with micelle Static Quenching Sometimes Stern-Volmer plots show a positive deviation from linearity for the highest concentrations of quencher used. These deviations are interpreted according to the sphere-of-action static quenching model. If a quencher is within a volume of V’ around the fluorophore, a quenching reaction occurs instantaneously after excitation with unit efficiency. Assuming that the quenchers are distributed among these volumes according to a Poisson distribution, the following expression for the combined existence of dynamic and static quenching is obtained I0 1 K SV QeV Q I KSV V dynamic quenching constant static quenching constant Static Quenching Example somatostatin in reverse micelle Example somatostatin in reverse micelle • Stern-Volmer plots are non-linear and exhibit upward curvature • Only some fraction of the excited states is actually quenched by the usual collisional mechanism. • Some of the excited states are deactivated statically after being formed by either an active volume element surrounding the fluorophoreor a dark complex between the reactant Example Fusarium solani Cutinase In native state the Trp fluorescence is highly quenched. Irradiation of the enzyme in the Trp absorption band causes an increase of fluorescence quantum yield. Steady State Emission Anisotropy Polarization Spectra The definition of the emission anisotropy P in a configuration where the exciting light is vertically polarized and the emitted fluorescence is observed at right angles in a horizontal plane P I|| and I are the polarized components parallel and perpendicular to the direction of polarization of the incident light, respectively. Steady State Emission Anisotropy Polarization Spectra Relationship between polarization and rotational correlation time 1 P 13 1 3 t t 1 1 P 3 0 0 t rot 4 r 3 3V kT RT P0 is the maximum P when the rotational motion is very slow trot rotational correlation time solution viscosity V= 4/3 r3N0= molar volume Resonance Energy Transfer • Energy transfer is the non-radiative transfer of energy from a donor to an acceptor. For energy transfer to occur the absorption spectra of the acceptor must overlap the emission spectra of the donor. Emission of D quenched Emission of A sensitized • The overlap is required such that quantum energy levels of equivalent energy exist for productive dipolar coupling between the two molecules. the rate of transfer depends on the distance between the donor and acceptor, energy transfer can be used to measure distances between sites on biopolymers. The distance range is much larger (20-70A) than possible with other spectroscopic techniques (e.g. NMR - 5 A), thus making fluorescence energy transfer a very useful technique. Resonance Energy Transfer • FÖrster Theory 1 kT t D R0 R 6 kT rate of energy transfer tD life-time of D in the absence of A R0 characteristic transfer distance~10-50 Å • Efficiency of Energy Transfer E kT /(kT 1/ t D ) 6 R0 6 /(R0 R ) 6 Fluorescence Spectroscopy Part III. Time-Resolved Fluorescence Measurements Time-Resolved Fluorescence Spectra Example Cutinase 20 oC I F t A B exp k r t C exp k d t IF(t) intensity of fluorescence measured at time t kr rate constant for the rise of the fluorescence intensity kd rate constant for the decay of the fluorescence intensity 40 oC Pulse Fluorimtetry For single exponential decay I t exp t / t For multi-exponential decay I t N exp t / t i i 1 i Obtain Decay curve of the fluorescence intensity after pulse excitation Fit the experimental decay curve With multi-exponential function Judge the fit with c (close to 1) W (randomly distributed around 0) Quality of the fit In the least-squares method, the first criterion is the reduced cr2 whose value should be close to 1 for a good fit. weighted residuals, If the fit is good, the weighted residuals are randomly distributed around zero. Decay Curve Decay of the fluorescence intensity (348.8 nm)after pulse excitation (302 nm) of 10mM cutinase Fresh Irradiated Example Conformations and Orientations of Aromatic Amino Acid Residues of Tachyplesin I in Phospholipids Membranes In the absence of liposomes In the presence of liposomes Tachyplesin I [Phe8]-Tachyplesin I [Phe13]-Tachyplesin I Excitation wavelength 295 nm in buffer in liposomes •Since tachyplesins are small the fluorescence anisotropy are small in buffer solution •In membrane, large increases in anisotropy values were observed Quality of the fluorescence decay analysis Tachyplesin I in Tris buffer Ex: 295 nm; em: 350 nm One-exponential Double-exponential Triple-exponential Quadruple-exponential Fluorescence decay function I , t 3 exp t / t i i i 1 Associated Emission spectra with individual decay time component (DAS) I i I ss f i f i i t i / t i i In Tris buffer In neutral DMPC In acidic DMPC Example Solvent-Exposed Tryptophans Probe the dynamics at Protein Surfaces Subtilisin Carlsberg Subtilisin Carlsberg Myelin basic protein