Dmitri Toptygin

Effects of the Solvent Refractive Index and Its Dispersion on the Radiative Decay Rate and Extinction Coefficient of a Fluorescent Solute

It is well known that the probabilities of radiative transitions in a medium differ from those in vacuum. Excitation of a fluorescent molecule and its radiative decay are examples of radiative transitions. The rates of these processes in solution depend on the optical characteristics of the solvent. In this article the radiative decay rate and the extinction coefficient of a fluorescent molecule in solution are expressed in terms of the intrinsic properties of the fluorescent molecule (electronic transition moments) and the optical characteristics of the solvent (refractive index, group velocity of light). It is shown that the group velocity does not enter in the final expressions for the radiative decay rate and the extinction coefficient; this means that the dispersion of the refractive index has no effect on these quantities. The expressions for both the radiative decay rate and the extinction coefficient contain the refractive index of the solvent and the local field correction factor. The latter depends on the cavity model, and, for some cavity models, on the shape of the cavity. Four types of cavity models are discussed; for each model the limits of applicability are examined. Experimental evidence in support of specific cavity models is reviewed.

Resolution of absorption spectra of rhodamine 6G aggregates in aqueous solution using the law of mass action

Abstract There is striking dissimilarity among the absorption spectra of rhodamine dimers reported by different authors. To address this issue, we measured the absorption spectra of aqueous solutions of rhodamine 6G over a wide range of concentrations. The lack of an isosbestic point near 508 nm indicates the presence of at least three species. Data analysis using the program spectrabind described in [D. Toptygin, L. Brand, Anal. Biochem. 222 (1995) 330] confirms the presence of trimers and tetramers in addition to dimers and monomers. The absorption peaks of the monomer, dimer, trimer, and tetramer are at 526, 498, 485, and 475 nm, respectively.

Spectrally- and time-resolved fluorescence emission of indole during solvent relaxation: a quantitative model

Abstract Relaxation of polar solvents around excited fluorescent solutes results in a continuous time-dependent red shift in the fluorescence spectrum. Static heterogeneous broadening can also produce time-variant fluorescence spectra. The two effects can be distinguished by a quantitative model of fluorescence during homogeneous electrostatic relaxation. The model describes the evolution of the instantaneous spectrum and the time variation of the intensity observed at any fixed wavelength. The model is in good agreement with the experimental data obtained using a solution of indole in anhydrous glycerol at 4 nm spectral and 65 ps temporal resolution.

Fluorescence decay and depolarization in membranes

In optically discontinuous and/or anisotropic environments such as lipid membranes, the radiative decay rate is dependent on the orientation of the emission dipole. A theory of fluorescence decay and depolarization in membranes has been developed that takes the orientational dependence into account. The theory predicts nonexponential total emission intensity decay and emission anisotropy decay different than the one in optically isotropic systems. 1,6‐diphenyl‐1,3,5‐hexatriene fluorescence in L‐α‐dipalmitoyl‐phosphatidylcholine large unilamellar vesicles has been analyzed in terms of this theory. It has been shown that the orientational dependence of the radiative rate is sufficient to account for the complexity of the intensity decay in membranes. The analysis also allowed the determination of the refractive index, orientational order, and rotational dynamics in the membrane.

Picosecond protein dynamics: the origin of the time-dependent spectral shift in the fluorescence of the single Trp in the protein GB1.

How a biological system responds to a charge shift is a challenging question directly relevant to biological function. Time-resolved fluorescence of a tryptophan residue reflects protein and solvent response to the difference in pi-electron density between the excited and the ground state. In this study we use molecular dynamics to calculate the time-dependent spectral shift (TDSS) in the fluorescence of Trp-43 in GB1 protein. A new computational method for separating solvent, protein, and fluorophore contributions to TDSS is applied to 100 nonequilibrium trajectories for GB1 in TIP3P water. The results support several nontrivial conclusions. Both longitudinal and transverse relaxation modes of bulk solvent contribute to the TDSS in proteins. All relaxation components slower than the transverse relaxation of bulk solvent have significant contributions from both protein and solvent, with a negative correlation between them. Five exponential terms in the TDSS of GB1 are well separated by their relaxation times. A 0.036 ps term is due to both solvent (60%) and protein (40%). Two exponential terms represent longitudinal (tau(L) approximately = 0.4 ps) and transverse (tau(D) approximately = 5.6 ps) relaxation modes of TIP3P water. A 131 ps term is attributable to a small change in the tertiary structure, with the alpha-helix moving 0.2 A away from the beta-strand containing Trp-43. A 2580 ps term is due to the change in the conformation of the Glu-42 side chain that brings its carboxyl group close to the positively charged end of the excited fluorophore. Interestingly, water cancels 60% of the TDSS resulting from this conformational change.

Femtosecond Fluorescence Spectra of Tryptophan in Human γ-Crystallin Mutants: Site-Dependent Ultrafast Quenching

The eye lens Crystallin proteins are subject to UV irradiation throughout life, and the photochemistry of damage proceeds through the excited state; thus, their tryptophan (Trp) fluorescence lifetimes are physiologically important properties. The time-resolved fluorescence spectra of single Trps in human gammaD- and gammaS-Crystallins have been measured with both an upconversion spectrophotofluorometer on the 300 fs to 100 ps time scale, and a time correlated single photon counting apparatus on the 100 ps to 10 ns time scale, respectively. Three Trps in each wild type protein were replaced by phenylalanine, leading to single-Trp mutants: W68-only and W156-only of HgammaD- and W72-only and W162-only of HgammaS-Crystallin. These proteins exhibit similar ultrafast signatures: positive definite decay associated spectra (DAS) for 50-65 ps decay constants that indicate dominance of fast, heterogeneous quenching. The quenched population (judged by amplitude) of this DAS differs among mutants. Trps 68, 156 in human gammaD- and Trp72 in human gammaS-Crystallin are buried, but water can reach amide oxygen and ring HE1 atoms through narrow channels. QM-MM simulations of quenching by electron transfer predict heterogeneous decay times from 50-500 ps that agree with our experimental results. Further analysis of apparent radiative lifetimes allow us to deduce that substantial subpopulations of Trp are fully quenched in even faster (sub-300 fs) processes for several of the mutants. The quenching of Trp fluorescence of human gammaD- and gammaS-Crystallin may protect them from ambient light induced photo damage.

Characterization of fluorescence quenching in bifluorophoric protease substrates.

NorFES is a relatively rigid, bent undecapeptide which contains an amino acid sequence that is recognized by the serine protease elastase (AspAlaIleProNle downward arrow SerIleProLysGlyTyr ( downward arrow indicates the primary cleavage site)). Covalent attachment of a fluorophore on each side of NorFESs elastase cleavage site enables one to use a change of fluorescence intensity as a measure of enzymatic activity. In this study two bichromophoric NorFES derivatives, D-NorFES-A and D-NorFES-D, were prepared in which D (donor) was tetramethylrhodamine and A (acceptor) was rhodamine-X, two chromophores with characteristics suitable for energy transfer. Absorption and fluorescence spectra were obtained with both the intact and cleaved homodoubly, heterodoubly and singly labeled derivatives. It was found that both the homo and hetero doubly-labeled derivatives form ground-state complexes which exhibit exciton bands. The hetero labeled derivative exhibits little or no resonance energy transfer. Spectral measurements were also done in urea, which partially disrupts ground-state dimers.

Fluorescence decay of DPH in lipid membranes : influence of the external refractive index

The radiative decay rate of a fluorescent probe in an optically thin layer is known to depend on the orientation of the probe and on the refractive indices inside and outside the layer (W. Lukosz, Phys. Rev. B 22 (1980) 3030). Fluorescent probes in phospholipid bilayer membranes approximate such a system. The natural lifetime is expected to vary with the refractive index of the medium surrounding the bilayer. The lifetime variation with the refractive index depends on the orientation of the fluorescent probe. This can be used to retrieve the second-rank orientational order parameter, . The fluorescence decay of all-trans 1,6-diphenyl-1,3,5-hexatriene in L-alpha-dipalmitoyl-phosphatidylcholine large unilamellar vesicles (LUVs) was measured at a temperature well below that of the phase transition. The refractive index of the medium was varied by addition of glycerol or sucrose. The observed change of decay time with the refractive index followed the theoretical prediction. The value of the order parameter, , recovered is significantly lower than that obtained from fluorescence polarization data. Possible reasons for this disagreement are discussed.

Determination of DPH Order Parameters in Unoriented Vesicles

This article reviews the determination of orientational order parameters in non-macroscopically oriented membranes from the data obtained with the fluorescent probe all-trans-1,6-diphenyl-1,3,5-hexatriene (DPH). Special attention is paid to the effect of microheterogeneity in the probe environment on the recovered values of the order parameters. An effort is made to accommodate new findings in the existing picture of orientational order in membranes.

The Identification of Tryptophan Residues Responsible for ATP-Induced Increase in Intrinsic Fluorescence of Myosin Subfragment 1

Abstract ATP binding to myosin subfragment 1 (S1) induces an increase in tryptophan fluorescence. Chymotryptic rabbit skeletal S1 has 5 tryptophan residues (Trp113, 131, 440, 510 and 595), and therefore the identification of tryptophan residues perturbed by ATP is quite complex. To solve this problem we resolved the complex fluorescence spectra into log-normal and decay-associated components, and carried out the structural analysis of the microenvironment of each tryptophan in S1. The decomposition of fluorescence spectra of S1 and S1-ATP complex revealed 3 components with maxima at ca. 318, 331 and 339–342 nm. The comparison of structural parameters of microenvironment of 5 tryptophan residues with the same parameters of single- tryptophan-containing proteins with well identified fluorescence properties applying statistical method of cluster analysis, enabled us to assign Trp595 to 318 nm, Trp440 to 331 nm, and Trp113, 131 and 510 to 342 nm spectral components. ATP induced an almost equal increase in the intensities of the intermediate (331 nm) and long-wavelength (342 nm) components, and a small decrease in the short component (318 nm). The increase in the intermediate component fluorescence most likely results from an immobilization of some quenching groups (Met437, Met441 and/or Arg444) in the environment of Trp440. The increase in the intensity and a blue shift of the long component might be associated with conformational changes in the vicinity of Trp510. However, these conclusions can not be extended directly to the other types of myosins due to the diversity in the tryptophan content and their microenvironments.