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Fluorescence spectroscopy of a green fluorescent protein mutant at single-molecule resolution has revealed a remarkable oscillatory behavior that can also be driven by applied fields. We show that immediately before unfolding, several periodic oscillations among the chemical substates of the protein chromophore occur. We also show that applied alternating electric or acoustic fields, when tuned to the protein characteristic frequencies, give rise to strong resonance effects.

The chemical unfolding behavior of porcine beta-lactoglobulin (PLG) has been followed at pH 2 and 6 in the presence of guanidinium hydrochloride. The PLG unfolding transition, monitored by tryptophan fluorescence, far and near UV circular dichroism and 1D-NMR, can be described by a three-state transition suggesting the presence of at least one intermediate state that appears to display an excess of non-native a-helical structures. The thermodynamic parameters, as determined through a global analysis fitting procedure, give estimates of the free energy differences of the transitions connecting the native, the intermediate and the unfolded state: DeltaG(NI)(0) = 2.8 +/- 0.7 kcal mol(-1) (pH 2) and 4.2 +/- 0.5 kcal mol(-1) (pH 6) and DeltaG(NU)(0) = 7.2 +/- 0.6 kcal mol(-1) (pH 2) and 6.9 +/- 0.6 kcal mol(-1) (pH 6). CD unfolding data of the bovine species (BLG) have been collected here under the same experimental conditions of PLG to allow a careful comparison of the two beta-lactoglobulins. Intermediates with different characteristics have been identified for BLG and PLG, and their nature has been discussed on a structural analysis basis. The thermodynamic data reported here for PLG and BLG and the comparative analysis with data reported for equine beta lactoglobulin, show that homologous beta-barrel proteins' belonging to the same family and displaying high sequence identity (52-64%) populate unfolding intermediates to different extents, even though a common tendency to the formation of non-native alpha-helical intermediates, can be envisaged. The present results provide a prerequisite foundation of knowledge for the design and interpretation of future folding kinetic studies. (C) 2004 Wiley-Liss, Inc.

The use of spectroscopy in the study of fatty acids binding to bovine beta-lactoglobulin (BLG) appears to be a difficult task, as these acid compounds, assumed as the protein natural ligands, do not exhibit favorable optical response such as, for example, absorption or fluorescence. Therefore, the BLG fatty-acid equilibrium has been tackled by exploiting the competition between fatty acids and ANS, a widely used fluorescent hydrophobic probe, whose binding sites on the protein have been characterized recently. Two lifetime decays of the ANS-BLG complex have been found; the longer one has been attributed to the internal binding site and the shorter one to the external site. At increasing fatty acids concentration, the fractional weight associated with ANS bound to the internal site drops, in agreement with a model describing the competition of the dye with fatty acids, whereas the external site occupancy appears to be unaffected by the fatty acids binding to BLG. This model is supported by docking studies. An estimate of the acid-binding affinities for BLG has been obtained by implementing the fitting of the bound ANS intensities with a competitive binding model. A relevant dependence has been found upon the solution pH, in the range from 6 to 8, which correlates with the calyx accessibility modulated by the conformation of the EF loop. Fatty acids with longer aliphatic chains (palmitate and laurate) are found to display larger affinities for the protein and the interaction free energy nicely correlates with the number of contacts inside the protein calyx, in agreement with docking simulations.

We have studied the fluorescence emission by two-photon excitation of four dyes widely used for bioimaging studies, rhodamine 6G, fluorescein, pyrene and indo-1 at the single molecule level. The single dye molecules, spread on a glass substrate by spin coating, show a constant fluorescence output until a sudden transition to a dark state very close to the background. The bleaching time that is found to vary in the series pyrene, indo-1, fluorescein and rhodamine 6G from the fastest to the slowest one respectively, has a Gaussian distribution indicating that the observed behavior is not due to photobleaching. Moreover, the bleaching time decreases with the glass substrate temperature reaching a vanishing nonmeasurable value for a limiting temperature whose value is found in the same series as for the bleaching time, from the lowest to the highest temperature respectively. The observed bleaching shows a clear correlation to the amount of absorbed power not reirradiated as fluorescence and to the complexity of the molecule. These observations are interpreted as thermal bleaching where the temperature increase is induced by the two-photon absorption of the single dyes as confirmed also by numerical simulations.

We have used the fluorescence anisotropy (FA) decay of retinol bound to bovine beta-lactoglobulin to monitor the time evolution of protein aggregation during the early stages of crystal growth. With this approach we have followed the formation of aggregates at different concentrations of ammonium sulfate, the precipitant used for crystallization. The average aggregation number is found to depend on precipitant concentration, and to be restricted to small numbers ranging from 2 to 5, also in the presence of visible growing crystals. The effect of particle distribution and of low probe-to-protein saturation on the FA response is also discussed in detail.

We investigate possible trapping effects induced by the infrared radiation employed for two-photon fluorescence spectroscopy and microscopy on the diffusion of small fluorophores and draw a compari...

The denaturation of beta-lactoglobulin (BLG) in the presence of urea and GuHCl has been investigated at different pH values with various spectroscopic techniques. The equilibrium denaturation free energy values, obtained by linearly extrapolating the data to vanishing denaturant (DeltaG(D)(H2O),), are compared and discussed. The fit of the spectroscopic data monitoring the denaturation oF BLG has been approached, at first, with a two-state model that describes the protein transition from the folded state (at each pH and in the absence of denaturant) to the denatured state. but in particular, along the GuHCl denaturation pathway some evidence is found of the presence of an intermediate state. Time-resolved fluorescence experiments performed on the BLG-ANS (1-anilino-8-naphtilalenesulfonate) complex help to understand the results. Fluorescence polarization anisotropy (FPA) measurements accompanying the denaturation process show the presence of a fast rotational diffusion of the ANS probe, and the data are interpreted in terms of local fluctuations of a still structured tract of the denatured protein where the probe is bound.

The fluorescence time decay parameters of the beta-lactoglobulin-1-anilinonaphthalene-8-sulfonate complex have been investigated under physical and chemical perturbations (2 < pH < 8 and added electrolyte 0 < NaCl < 0.5 M) to obtain new insight on the nature of the protein binding interactions. A double exponential decay of the bound probe lifetime has been confirmed by the presence of a longer component, 11 to 14.5 ns, and a shorter component, 2.5 to 3.5 ns. The two lifetimes are ascribed to different binding modes associated also with different exposure to the solvent; in particular, the longer component is attributed to binding inside the hydrophobic beta barrel, while a "surface" site is suggested for the shorter component. A detailed analysis of the lifetime fractional intensities correlates the binding constants with ionic strength and supports the presence of electrostatic effects at both sites. A Debye-Hückel approach, applied to extrapolate the electrostatic free energy contribution vs. pH at vanishing ionic strength, gives interesting clues on the effective charge felt by the ANS ligands in the proximity of each site. In particular, binding is found to parallel the aspartate and glutamate titrations between pH 3 and pH 4.5; the "surface" site mainly responds to the presence of these local titrating charges while the "internal" site more closely follows the overall protein net charge.

β-Lactoglobulin, a protein belonging to the lipocaline family, is studied by Photon correlation spectroscopy and small angle X-ray scattering in acidic solutions (pH = 2.3) and at ionic strengths in the range 7−507 mM. Both experiments give a clear evidence of a protein monomer−dimer equilibrium affected by the ionic strength of the solution. The interparticle interactions are determined by means of Photon correlation spectroscopy by following the trend of the protein diffusion coefficient versus concentration (from 0.2 to 25 g/L) at ionic strengths of 7 and 107 mM. From a detailed analysis, estimates of the protein charge, hydrodynamic radius, and dimeritazion fraction have been obtained. Small angle X-ray scattering measurements performed versus ionic strength (from 7 to 507 mM) in dilute solutions (10 g/L) allow an independent estimate of the dimerization fraction. A global fit of the SAXS scattered intensities at different salt concentrations using a simple electrostatic model for the aggregation mech...

The average diffusion coefficients of hemoglobin (bovine CO-Hb) in dilute solutions (φ = 0.005), as obtained by cumulant analysis of the autocorrelation functions of the scattered light, are found to depend upon pH and ionic strength. A decrease of the diffusion coefficient is observed vs ionic strength for two salts, NaCl or CaCl2, in the range 10−300 mM, when the solution pH is away from that corresponding to the isolectric point of the protein. It is shown here that the results can be interpreted in terms of electrostatic effects with a model that should include both interparticle interactions and subunit dissociation of the protein. Global analyses of the first cumulant diffusion coefficient vs ionic strength were then applied in order to obtain accurate values of the hemoglobin charge vs pH and of its single particle diffusion coefficient. A quantitative estimate of the dissociation was also obtained, leading then to good descriptions both of the electrostatic and of the non-electrostatic contributio...

Experimental and theoretical analysis of the effect of both intermolecular interactions and dissociation on the diffusional properties of carbon–monoxide bovine hemoglobin in solution are reported here. When performing accurate photon correlation spectroscopy measurements versus protein concentration, even on dilute solutions, one finds that the first cumulant diffusion coefficient of the macromolecule has a relevant dependence upon pH (5⩽pH⩽9.5), ionic strength (10–100 mM), and, particularly, on hemoglobin concentration (1⩽c⩽20 mg/ml). The results cannot be interpreted by considering the occurrence of either protein dissociation or intermolecular interactions only. As a consequence a simple theoretical expansion of the first cumulant diffusion coefficient, to first order in concentration, is derived here with the inclusion of protein interactions and dissociation. A fit procedure based on this expression leads to a good description of the dissociation and an accurate estimate of the native protein charge...

The conformation and slow internal motions of a DNA plasmid molecule pUC18 (2687 base pairs) are studied by means of depolarized photon correlation spectroscopy. The autocorrelation functions are measured at different scattering angles between 6.7° and 90° and analyzed as heterodyne autocorrelation functions. One obtains both a relaxation time of ≂140 μs, which is interpreted as the tumbling rotational diffusion coefficient of the plasmid, and some faster component with relaxation time around 15 μs. The value of the rotational diffusion relaxation time is in good agreement with the results of Monte Carlo simulations, while the faster decay has a nonsingle exponential decay behavior. The results of measurements made in the depolarized scattering configuration are compared to those obtained from polarized configuration and critically discussed.

Bovine carbonmonoxy hemoglobin investigated with light scattering studies is found to dissociate from its native tetramer structure into dimers and monomers. The values of the hydrated tetramer radius, RT = 32.1 A, and the fractional dissociation vs pH, have been obtained at different ionic strengths from the autocorrelation function of the scattered light. The results suggest that a relevant contribution to Hb dissociation is due to electrostatic effects and, by means of a model derived by Tanford, it has been possible to predict the behavior of dissociation. Among the findings of this approach, we recall the estimates of the electrostatic energy contributions to Hb dissociation, up to ≃ GRT, and the predicted charge of tetrameric Hb vs pH, which agrees very well with the experimental data. © 1994 John Wiley & Sons, Inc.

Dynamic light scattering from protein solutions can be applied to the detection of conformational changes and to the measurement of particle size. Because accurate results can be adversely affected by experimental perturbations, careful procedures are necessary both in data acquisition and in data analysis. Autocorrelation functions (ACF?s) of scattered light are simulated here to evaluate the role of the perturbations affecting measured signals from dilute protein solutions. The analysis of measured and simulated ACF?s has been performed both by cumulant expansion and by a nonlinear least-squares fit, thereby allowing the definition of criteria for the optimization of the fitting parameters and of the measuring conditions. Moreover, by comparing experimental data from hemoglobin solutions and computer simulations, we show how to evaluate the contributions of polydispersity and statistical noise affecting the measurements.

New probes for kinetic intracellular measurements in the millisecond range are desirable to monitor protein biochemical dynamics essential for catalysis, allosteric regulation and signalling. Good candidates to this aim are the photo-switchable mutants of the Green Fluorescent Protein, whose anionic fluorescence, primed by blue light, is markedly enhanced under an additional excitation at a shorter wavelength and relaxes within few milliseconds. The aim of this report is to study how the brightness enhancement kinetics depends on the physical-chemical and spectroscopic parameters and to provide proof-of-concepts experiments for the use of the fluorescence enhancement in conditions in which the protein diffusion is hindered and thereby photo-bleaching can be a limiting critical issue. Future, direct applications of photo-chromic mutants for modulated excitation imaging would in fact require such a detailed knowledge. We present here an extensive study of the photo-switching mechanism of the E222Q mutant of GFPMut2 (Mut2Q), pumped by visible 488 nm light and probed at 400-420 nm, as a function of pH, viscosity, temperature and light intensity. In solution, two characteristic photo-switching times are found by means of modulated double beam fluorescence correlation spectroscopy in the 1-30 ms range, depending on the solution pH. The photo-switching kinetics is solved in terms of the eigenvalues and the eigenvectors of a specific energy diagram and directly used to fit the data, suggesting that the observed photo-switching amplitudes and kinetics are related to a single three-levels transition loop. Finally we give in-vitro examples of the use of modulated excitation microscopy, based on fluorescence enhancement amplitude and kinetics detection, on Mut2Q protein samples immobilized in acrylamide gels.

The plasmid pUC18m containing the insertion of the curved sequence AluI, repeated from one to six times, has been studied by dynamic light scattering.

GFP mutants are known to display fluorescence flickering, a process that occurs in a wide time range. Because serine 65, threonine 203, glutamate 222, and histidine 148 have been indicated as key residues in determining the GFP fluorescence photodynamics, we have focused here on the role of histidine 148 and glutamate 222 by studying the fluorescence dynamics of GFPmut2 (S65A, V68L, and S72A GFP) and its H148G (Mut2G) and E222Q (Mut2Q) mutants. Two relaxation components are found in the fluorescence autocorrelation functions of GFPmut2: a 10-100 micros pH-dependent component and a 100-500 micros laser-power-dependent component. The comparison of these three mutants shows that the mutation of histidine 148 to glycine induces a 3-fold increase in the protonation rate, thereby indicating that the protonation-deprotonation of the chromophore occurs via a proton exchange with the solution mediated by the histidine 148 residue. The power-dependent but pH-independent relaxation mode, which is not affected by the E222Q and H148G mutations, is due to an excited-state process that is probably related to conformational rearrangements of the chromophore after the photoexcitation, more than to the chromophore excited-state proton transfer.

Two DNA plasmids, pEGF and pACL29, intercalated with ethidium bromide (EB), have been examined by circular dichroism (CD) and dynamic light scattering (DLS). CD and DLS data show significant changes when the EB/DNA (phosphates) ratio reaches a value of r approximately equal to 0.13. The translational and rotational diffusion coefficients, predicted assuming that plasmids can be described by a string of beads, and the CD spectrum, suggest that a transition from an interwound to a toroidal conformation is likely to occur.

We present evidence of conformational substates of a green fluorescent protein mutant, GFPmut2, and of their relationship with the protein behavior during chemical unfolding. The fluorescence of single molecules, excited by two infrared photons from a pulsed laser, was detected in two separate channels that simultaneously collected the blue or the green emission from the protein chromophore chemical states (anionic or neutral, respectively). Time recording of the fluorescence signals from molecules in the native state shows that the chromophore, an intrinsic probe sensitive to conformational changes, switches between the two states with average rates that are found to assume distinct values, thereby suggesting a multiplicity of protein substates. Furthermore, under denaturing conditions, the chromophore switching rate displays different and reproducible time evolutions that are characterized by discrete unfolding times. The correlation that is found between native molecules’ switching rate values and unfolding times appears as direct evidence that GFPmut2 can unfold only along distinct paths that are determined by the initial folded substate of the protein.

The chromophore of a green fluorescent protein (GFP) mutant engineered to enhance emission and stability is known to display erratic switchings among a few of its chemical substates and, in particular, between the anionic A and the neutral N substates, whose difference is associated with a proton exchange and a consequent conformation rearrangement. However, when close to unfolding, the A-N switchings suddenly become very regular as shown by fluorescence oscillations that have been recently observed for molecules embedded in wet silica gel. In order to establish whether the matrix hosting the protein is responsible for these oscillations, we investigated the effect of another medium (silanized surfaces), of a different denaturant (urea) and of cosolvents (D2O and glycerol). The occurrence of periodic A-N switchings, in the last milliseconds before GFP unfolding, is observed under all investigated conditions, together with three specific frequency values that characterize the pre-unfolding fluorescence. Urea and guanidinium, the denaturants employed in order to unfold GFP, do not lead to appreciable differences in the observed switching parameters, whereas the different media embedding the protein give rise only to frequency shifts that scale with the viscosity of the host. The periodicity of the GFP A-N switchings and their dependence on cosolvents suggest that they could be associated with oscillatory motions between meta-stable conformations of the β-barrel surrounding the chromophore near protein unfolding. © 2007 EBSA.

An organic dye, SAMSA, bound to gold nanoparticles, displays random photoactivated fluorescence blinking whose rate depends on the size of the nanoparticles. We report experiments indicating that (1) the dye emission wavelength is red-shifted (10-30 nm) by applying an external low voltage (1-10 V) and that (2) the fluorescence emission of single dyes can be resonantly driven by tuning the alternating external bias frequency from 1 to 3 Hz, depending on the nanoparticle size. These properties appear highly valuable and promising for devising light emitting nanostructures. © 2007 American Chemical Society.

Dimethyl-pepep (D-pepep), a newly developed and very efficient two-photon absorber, has been tested here for two-photon excitation (TPE) cellular imaging. The spectral characteristics of the dye following one-photon excitation (OPE) and TPE (excitation and emission spectra, fluorescence lifetime, molecular brightness, saturation intensity) are reported. In vitro interaction studies with biomolecules show that dimethyl-pepep has a large affinity for DNA. A comparison with a widely used DNA stainer, 4-6-diamidino-2-phenylindole (DAPI) bound to DNA shows that the D-pepep brightness is one order of magnitude higher than that of DAPI, making this dye suitable for microscopy and imaging applications. TPE images taken from double-stained yeast Saccharomyces cerevisiae cells have revealed that D-pepep localizes mainly in the nucleus, similarly to DAPI, and in mitochondria, although to a minor extent. Preliminary tests have shown that the dye cellular toxicity is negligible. © 2004 Elsevier B.V. All rights reserved.

Fluorescence anisotropy decay spectroscopy is a suitable tool for investigating the size and the shape of biological molecules. We coupled this technique to an optical microscope in order to reduce the excitation volume and to allow its application to spatially inhomogeneous samples. Phase modulated measurements of the fluorescence anisotropy decay were performed by feeding an intensity modulated linearly polarized laser beam to the epifluorescence port of a microscope. Here we report the test of the dynamic response of the microscope by comparing the lifetime and fluorescence polarization anisotropy decays obtained in cuvettes in a standard phase modulation fluorometer and on tiny drops on the microscope stage. We show that once a correction factor for the objective depolarization is introduced in the best-fit functions for the data analysis of the decays, the results obtained on the two setups are comparable. Some applications are reported here on long DNA tracts as well on short DNA fragments containing structural anomalies.

Confocal microscopy is one of the most widely used and non-invasive tool for the investigation of biological matter. It improves the performances of the optical microscope by reducing the excitation volume and enhancing the axial resolution due to the use of high numerical aperture lenses. We have adapted an inverted confocal optical microscope to the measurement of fluorescence emission dynamics (lifetime and fluorescence polarization anisotropy). The dynamic spectroscopy measurements are obtained with phase fluorometry and are based on a modulated linearly polarized laser beam which is fed to the epifluorescence port of the microscope. We report the test of the microscope by comparing the lifetime and fluorescence polarization anisotropy decay obtained in cuvettes in the standard phase modulation fluorometer and on tiny drops on the microscope stage. We show that once a correction factor is introduced in the best fit functions used in the data analysis of the decays, the results obtained on microliters volumes is comparable to those obtained in cuvettes in the standard phase fluorometer spectrometer. An example of application is reported on short DNA fragments.

The stabilizing role of the disaccharide trehalose on beta-lactoglobulin (BLG) against its chemical denaturation both at native and acidic pH has been explored by means of time-resolved fluorescence of the probe acrylodan covalently bound to the unique free cysteine of BLG. The changes in acrylodan fluorescence lifetime with guanidinium chloride concentration reveal BLG sigmoidal denaturation profiles which depend upon the amount of trehalose in solution. When adding trehalose the transition midpoint shifts towards higher denaturant concentration. This effect has been measured by fitting the data with a two-state model whose parameters indicate that an almost 60% increase in the denaturation free energy is induced independently of trehalose concentrations and pH values. Fluorescence anisotropy measurements performed in the same conditions reveal that the internal dynamics are largely affected by the sugar, which makes the acrylodan environment more rigid, and by the denaturant that acts in the opposite way. The overall rotational diffusion of BLG suggests that trehalose affects the hydrodynamic properties of the solution in the proximity of the protein; tentative mechanisms are discussed.

Single molecule spectroscopy often requires the immobilization of the molecules onto solid or quasi-solid substrates and the use of relatively high excitation intensity. We have studied the fluorescence emission of four common dyes used for bio-imaging studies, rhodamine 6G, fluorescein, pyrene and indo-1 at the single molecule level under two-photon excitation regime. We focus on two-photon excitation thermal effects on the stability of the single molecules, influencing the internal photo-dynamics and the total duration of the fluorescent emission. Single dye molecules, spread on a glass substrate by spin coating, show a constant fluorescence output till a sudden transition to a dark state. The bleaching time varies in the series pyrene, indo-1, fluorescein and rhodamine 6g from the fastest to the slowest one respectively, has a gaussian distribution suggesting that bleaching is not due to photo-bleaching. These observations are interpreted as thermal bleaching where the temperature increase is induced by the two-photon excitation process and the thermal bleaching is correlated to the amount of absorbed power that is not re-irradiated as fluorescence.

We report on the construction details of a compact autocorrelator set-up for the measurement of the width of infrared laser pulses at the focal plane of a microscope for two-photon excitation fluorescence imaging. One of the novelties of the set-up, which leads to an improved measurement accuracy, is the use of a modulation technique that is achieved by mounting one of the interferometer mirrors on a loudspeaker driven by a sinusoidal bias at low frequency. A non-linear least-square routine selects only that part of the fluorescence signal that is modulated at the same frequency as the loudspeaker bias. To further increase the accuracy, the laser pulse width is obtained from a series of measurements at different values of the modulation bias. The autocorrelator is a compact single bread-board (10 x 20 cm); it is PC-controlled both for the acquisition and the analysis of the data and can be coupled to different ports of the microscope. The increase in the pulse width measured for three different ports of the microscope is well accounted for by the group velocity dispersion and the glass thickness of the optics found along these paths.

The synthesis and characterization of novel heteroaromatic-based two-photon absorption (TPA) dyes for bio-conjugation is described; the new isothiocyanate and maleimide derive from a class of novel efficient quadrupolar and octupolar/branched chromophores relying on the electronic effects of electron-poor and electron-rich simple heteroaromatic rings; the new systems exhibit very large TPA cross-sections, high chemical stability, and very low photobleaching.

The Ku heterodimer plays a central role in non-homologous end-joining. The binding of recombinant Ku to DNA has been investigated by dynamic light scattering, double-filter binding, fluorescence spectroscopy, and band shift assays. The hydrodynamic radius of Ku in solution is 5.2 nm and does not change when a 25-bp double-strand DNA (dsDNA) fragment (D25) is added, indicating that only one Ku molecule binds to a 25-bp fragment. The dissociation constant (k(d)) for the binding to D25 is 3.8 +/- 0.9 nm. If both ends of the substrate are closed with hairpin loops, Ku is still able to bind with little change in the k(d). The k(d) is not affected by ATP, Mg(2+), or ionic strength. However, the addition of bovine serum albumin decreases the k(d) by 2-fold. DNA substrates of 50 bp can bind two Ku molecules, whereas three molecules are bound to a 75-bp substrate. Data analysis with the Hill equation yields a value of the Hill coefficient (n) close to 1, and the k(d) values for the binding of Ku to both ends of these substrates are the same. Thus, we demonstrate that there is no cooperative interaction among the Ku heterodimers binding longer substrates.

We have combined a confocal laser scanning head modified for TPE with some spectroscopic modules to study single molecules and molecular aggregates of rhodamine 6G and labeled proteins on glass substrates. The fluorescence intensity of the spots occurs at definite values that are multiples of a reference signal and extrapolate to the background level measured on the images. These properties suggest that these spots arise from single rhodamines. The discrete character of the intensity distribution can therefore be used as a simple, quantitative tool to discriminate between single molecules and molecular aggregates on single snapshots. These studies have been performed by using a combined confocal and spectroscopic architecture realized for two-photon excitation.

We have characterized a commercial confocal scanning head for the detection of single molecule fluorescence by two-photon excitation. We have verified that the distribution of the fluorescence emitted by dyes and labeled proteins on glass substrates is discrete with quanta proportional to a common reference signal. We describe and test a simple and quantitative tool to discriminate between single molecules and molecular aggregates on single snapshots based on the analysis of the intensity distribution. We have verified the square dependence of the fluorescence intensity vs. the excitation power, suggesting that no appreciable saturation and fast photo-damage of the chromophores takes place at the excitation power employed here. Microsc. Res. Tech. 55: 359 -364, 2001. © 2001 Wiley-Liss, Inc.

Steady-state and dynamic fluorescence titrations show that: (a) the complex between beta-lactoglobulin (BLG) and 1-anilinonaphthalene-8-sulfonate (ANS) displays a heterogeneous equilibrium with large changes in the binding strength vs. pH and ion concentration; and (b) the fluorescence response of bound ANS reveals two separate lifetimes that suggest two different sites (or binding modes). While steady-state fluorescence titrations yield effective values of the binding constant and of the bound ANS quantum efficiency, it is shown that, by combining steady-state fluorescence and lifetime decay of ANS, it is possible to give quantitative estimates of the association constants for each site. When heading from the acid (pH approximately 2) to the native state (pH approximately 6) the main result is a very large reduction of the effective binding constant. This and the results of titrations vs. ionic strength suggest that electrostatic interactions are a major contribution to ANS binding to BLG.

The dependence of the spinning diffusion coefficient of a wormlike chain upon contour length L, persistence length P, and radius R is shown here to follow a "Lorentzian" law of width Γ vs σβL/R, where σβ2=l0/P is the variance of the bending angles distribution of Monte Carlo simulated chains with bond length l0. This description is equivalent to that of a spinning cylinder of length L and effective radius Reff=R(L,P), with Reff≥R. When considering experimental data it is found that fluorescence polarization anisotropy (FPA), a technique very sensitive to spinning, also yields apparent DNA radii depending upon fragment length. In order to derive DNA parameters which are independent of fragment length, we introduce a procedure for fitting FPA data which takes into account thermal distortions and employs the parametric expressions for rigid body rotations, spinning and tumbling, depending only upon L, P, and the actual DNA radius, R. Then the apparent persistence length P can be estimated once a value of R is assumed together with the value of the dynamic persistence length, the latter affecting the internal bending motions of the fragments. Fitting the FPA data is easily accomplished with the value of R = 10 Å as suggested by a number of recent measurements. © 1996 American Institute of Physics.

The decay of the fluorescence polarisation anisotropy (FPA) of the ethidium-DNA complex has been measured by multifrequency phase fluorometry, in order to study the perturbations induced by the presence of different ligands on the torsional dynamics of DNA, a moderately flexible polymer that undergoes bending (flexure of the helix axis) and torsional (twisting of base pairs) motions. Two probes have been used together with ethidium: an intercalator, chloroquine, and a minor groove binding dye: hoechst 33258. Chloroquine is found to substantially modify the DNA torsional dynamics both in linear and in circularly closed DNAs only at high binding ratios, in agreement with previous reports [Wu et al. Biochem. 27 (1988) 8128]. The effective elastic constant becomes approximately three times larger when the dye/base pairs binding ratio is higher than 0.14. The minor groove ligand hoechst 33258, on the other hand, greatly increases the effective elastic constant to the point that at dye/base pairs ratios larger than 0.5, the effective elastic constant becomes stiffer by several orders of magnitude, suggesting a progressive hindering of internal motions. The results reported here show that DNA torsions are more effectively influenced by groove-binding molecules than by intercalators and it is expected that the large perturbation of the former ligand may be useful when describing the change in the dynamical properties induced by DNA binding proteins. FPA in the frequency domain, the technique adopted throughout this work, has proved to be very sensitive to changes of the elastic constant that describes DNA torsional dynamics. Several computer simulations performed in order to predict the FPA time decay of intercalated ethidium have led to good agreement with the observed results. © 1995.

The technique of fluorescence polarization anisotropy (FPA) decay of intercalated ethidium has been used to study DNA conformation and dynamics, which are being recognized as primary determinants in transcription control and other cellular processes. Frequency modulated FPA when applied to two DNA molecules, a "straight" 50 base-pairs duplex fragment, and a bent fragment of similar length, has yielded different rotational diffusion coefficients for the two fragments. The data have been processed with an analytical model and with Brownian dynamics simulations, obtaining a good fit and a quantitative agreement between the two models. Both analyses have confirmed that one fragment can be described as a straight cylinder, while the other fragment is bent, with an angle estimated to be 45 degrees +/- 3 degrees. FPA has proved to be very powerful in determining simple conformations of short DNA duplexes and also particularly apt to probe the dynamical features of DNA fragments where conventional methods are either too cumbersome or fail to give quantitative results. In addition, the ligand no longer behaves ideally due to its complex structure and charge distribution. Thus for the protein the slope is no longer related simply to the net ligand charge, and the PB model gives a much larger slope than the LL model.(ABSTRACT TRUNCATED AT 400 WORDS)

The effect of the insertion of different amounts (from 0 to 6) of the curved sequence AluI in pUC18m plasmid (2686 base pairs, bp) is studied by dynamic light scattering. This sequence is a highly repeated 113 base pairs long sequence from Artemia Franciscana shrimp. A 30% compaction of the plasmids containing 2 and 6 adjacent AluI sequences compared to pUC8 plasmid (2717 bp) is observed. Furthermore the behavior of the translational diffusion coefficient Dt versus the number of adjacent AluI insertion is not monotonic.

The time decay of the fluorescence polarization anisotropy of calf thymus DNA-ethidium complexes is obtained from measurements with sine-modulated excitation employing the so-called multifrequency phase fluorometry. A torsional dynamics model developed by J. M. Schurr [(1984) Chemical Physics, Vol. 84, pp. 71–96] and translated into the frequency domain is found here to describe accurately DNA-ethidium fluorescence data collected under modulated excitation. At a low dye/DNA ratio (1 : 400) the value of the DNA torsional constant (α = 4.63 ± 0.2 10−12 dyne cm) fitting the data agrees very well with the known values of α. When the measurements are extended to a higher ethidium/DNA ratio, energy transfer effects between intercalated dyes are observed. A theoretical prediction of the donor and acceptor dye contributions to the fluorescence polarization anisotropy is made here, taking into account also dye-dye distance distributions. © 1992 John Wiley & Sons, Inc.

The fluorescence lifetime of ethidium bromide (EB), a widely used fluorescent dye, has been monitored in water solution versus trehalose concentration in order to learn how the presence of the disaccharide modifies the hydration of EB, which is used in this study as a model probe. The interest in trehalose, a naturally occurring bioprotector (osmolyte), stems from its ability to stabilize biomolecules under stress situations. The observed effects of trehalose on EB fluorescence properties have been compared with those induced by sucrose and glucose. Lifetime measurements have been performed by frequency domain fluorometry (2-40 MHz), and from the analysis of their changes versus sugar concentration, the EB-sugar interaction parameters have been obtained. The effect of trehalose on the EB decay in glasses has also been studied both by exploring the heterogeneity of lifetime decay and by single-molecule imaging. Trehalose appears to be more efficient in changing the EB fluorescence parameters, such as the emission lifetime, and it leads to a degree of heterogeneity higher than that induced by the other sugars. When EB is embedded in trehalose glasses, the heterogeneity of the emission and of the bleaching time is further enhanced.

The binding of ethidium bromide to calf thymus DNA is investigated over a wide range of temperatures and alcohol concentrations by means of fluorescence techniques. The presence of alcohol is found to lower the association constant, as isopropanol and n-propanol are more effective than ethanol and methanol. It is also found that the data from different alcohols fall very close on the same straight line when plotted vs the solution dielectric constant ϵ. It is shown that the dependence of the binding constant on ϵ is beyond the prediction of current polyelectrolyte theories; rather, it appears that the binding is greatly controlled by the activity coefficient of the dye. The thermodynamic functions of the reaction have been estimated by means of Van't Hoff analysis: a large, negative enthalpy contributes to the binding, whereas the entropy, also favorable, is only slightly responsible for the overall free energy. Both enthalpy and entropy are strong functions of temperature and alcohol concentration. We show that the dependence of the thermodynamic quantities on the temperature is removed by considering “isodielectric” binding. This can be accomplished by a proper choice of the alcohol concentration, so that the thermal dependence of ϵ is compensated. It is found that the isodielectric free energy, enthalpy, and entropy are the same for different alcohols when plotted vs ϵ. When looking at the isodielectric quantities, it appears that the binding is now opposed by a positive enthalpy, whereas the observed favorable free energy results mainly from a large and positive entropy. The relevance of hydrophobic effects emerges from the behavior of the isodielectric thermodynamic quantities, as well as from the similarities observed when other interacting systems are compared.

The isothermal denaturation of calf thymus DNA, induced by the presence of some monohydric alcohols in the solution, was investigated. Measurements were performed at a temperature (67.2°C) at which the denaturation, in the absence of alcohols, is about 20% and melting profiles at varying temperatures were also recorded. Results show that with increasing alcohol concentration and alkyl group size DNA denaturation first reaches a maximum and then falls back. This behaviour, ascribed to «unusual» water properties as inferred also from compressibility measurements, indicates that hydrophobic and electrostatic effects are connected in a complex way.

The interaction of DNA with the dye ethidium bromide (EB) has been investigated under the presence of methanol at varying temperatures. The Gibbs free energy associated with the DNA-EB binding is found to be well described by its linear dependence upon the dielectric constant of the solution, a property also found for other alcohols. When the enthalpy and entropy associated with the binding are considered, it is found that their role becomes rather simple if their evaluation is performed isodielectrically (e=const). According to this procedure, the varying properties of the solvent can be separated from those of the interacting DNA and EB and the presence of hydrophobic interactions becomes then well established.

The binding of a few drugs (ethidium bromide, propidium diiodide, proflavine and actinomycin D) to DNA has been investigated in aqueous solutions to which cosolvents of different polarity have been added. It is found that both alcohols (less polar than water) and amides (more polar) lower the binding constant according to a linear relationship between the intercalation free energy and cosolvent concentration. The main action of cosolvents cannot be described in terms of electrostatic effects, since they predict much smaller changes in the binding constant than those observed. It appears instead that relevant solvation effects are responsible for the binding strength of the different dyes to DNA. As a general result, it is found that solvation effects largely contribute to the intercalation free energy, thereby weakening the influence of nonspecific interactions at the intercalation site.

The flourescence spectrum of ethidium, a well-known dye that binds DNA by intercalation, is interpreted in terms of vibronic components. The role of solvents such as water, ethanol and glycerol together with that of the host DNA is examined and the parameters of the linear electron-vibration interaction are estimated. Hard mode frequencies of 1200–1300 cm −1 and soft mode frequencies of 200–300 cm −1 are obtained. A description of the thermal dependence of the ethidium fluorescence quantum yield in different media between 100 and 350 K is attempted in terms of the thermal activation to a radiationless level.

The determination of equilibrium constants, seen,e.g., in the usual Scatchard plots, may be highly inaccurate because generally the values of the bound-ligand response are known with large uncertainty. A common cause of this inaccuracy stems from the use of samples that contain small amounts of free ligand, hardly avoided, or from equilibrium dialysis which is generally affected by the Donnan effect, dye losses, etc. From an analysis of this problem a simple procedure is derived here, so that, in spite of the above drawbacks, the equilibrium parameters can be obtained with better precision.

The affinity of quinacrine for native DNA has been determined from fluorescence measurements and equilibrium dialysis in Tris-HC10.05 m , NaCl0.1 m , EDTA 10 −3 m , pH 7.5. When considering M. lysodeiktikus, E. coli calf thymus and C. perfringens the affinities of DNA for quaniactive have been found to change by a factor of two and the fluorescence intensities to change by a factor of 25. The varying affinities and fluoroescence intensities of bound quinacrine are attributed to heterogeneous binding. For all DNAs we have assumed that there exist three classes of intercalation sites: I, A·T-A·T; 2, G·C-G·C; and 3, A·T-G·C, assuming that base pair ordering is less relevant than base composition of sites. By fitting the affinities of native DNAs with this model it was found that quinacrine binds to site 2 three times more strongly than it does to site 1. When flucrescence intensity is studied, triplets of A·T pairs appear to be responsible for the high quantum yield of A·T rich DNA whereas the quenching properties of a G·C base pair adjacent to an intercalated quinacrine are well known.

The experimental investigation in the temperature range from 4 to 400\ifmmode^\circ\else\textdegree\fi{}K of the ultraviolet $U$ band in KCl, KBr, and RbCl crystals containing ${\mathrm{H}}^{\ensuremath{-}}$ and ${\mathrm{D}}^{\ensuremath{-}}$ ions has revealed the following isotope effects: (a) The half-width of the ${U}_{\mathrm{H}}$ band is from 4 to 8% larger than that of the ${U}_{\mathrm{D}}$ band. (b) The ${U}_{\mathrm{H}}$ band occurs at energies \ensuremath{\sim}0.02 eV smaller than those of the ${U}_{\mathrm{D}}$ band. In order to describe these results the method of moments, in the harmonic and Condon approximations, has been applied. From the symmetry of the defect it is shown that only the vibrational modes which transform according to the ${{\ensuremath{\Gamma}}_{15}}^{\ensuremath{-}}$ representation of the full cubic point group can be responsible for the isotope effects; these modes are just those active in the infrared. It is found that during optical transitions, allowed in dipole approximation, the ${{\ensuremath{\Gamma}}_{15}}^{\ensuremath{-}}$ modes suffer, via the electron-phonon interaction, only a frequency shift, and that only the local mode has to be taken into account in order to predict the observed isotope effects, since the contribution coming from the ${{\ensuremath{\Gamma}}_{15}}^{\ensuremath{-}}$ continuum is negligible. Finally, the local-mode frequency of the excited $U$ center has been estimated.

The absorption spectra of all the alkali halides except LiF and LiI, measured in the range 5.0 to 12.0 eV at 10\ifmmode^\circ\else\textdegree\fi{}K, are presented. These data complement earlier measurements made at 80\ifmmode^\circ\else\textdegree\fi{}K. Identification of excition lines involving $d$-like electrons is made by a comparison of the crystal absorption and the absorption spectra of rare-gas atoms. From the positions of exciton states with $d$-like electrons, the energy of the second conduction band at $\ensuremath{\Gamma}$ is deduced for some of the alkali halides. The results of these deductions are compared with recent calculations of the conduction-band structure of KI and KCl.

The absorption spectra of thin films of argon, krypton, and xenon have been measured between 20 and 50\ifmmode^\circ\else\textdegree\fi{}K in the region from 1600 to 900 \AA{} (8 to 14 eV). The spectra show: (a) strong doublets corresponding to the atomic resonance doublets, (b) lines apparently of nonatomic nature, and (c) absorption continua. The experimental results are discussed in terms of the Frenkel and Wannier exciton models and the band gaps are estimated. Reasonable agreement is found between experimental results and available theoretical predictions.