Conformational differences of oxytocin and vasopressin as observed by fluorescence anisotropy decays and transient effects in collisional quenching of tyrosine fluorescence

We used gigahertz frequency-domain fluorometry to examine the tyrosyl fluorescence intensity and anisotropy decays of the single-tyrosine cyclic peptide hormones oxytocin and vasopressin. Acrylamide quenching and a distance-dependent quenching model for collisional quenching were used to evaluate the extent of tyrosyl exposure to the quencher and to provide increased resolution of the picosecond anisotropy decays. Analysis of the intensity decays using a lifetime distribution model shows different distributions for oxytocin and vasopressin. We found that the tyrosyl fluorescence of lysine-vasopressin, as revealed both by the lifetime Stern-Volmer plots and from the quenching analysis, is quenched more effectively than oxytocin. ForN-acetyltyrosinamide (NATyrA), oxytocin, and lysine-vasopressin, we recovered apparent diffusion coefficients for quenching of 4.7×10^–6, 0.44×10^–6, and 4.3×10^–6 cm²/s, respectively, the lower value for oxytocin suggesting a shielded environment for its tyrosyl residue. Tyrosyl anisotropy decays were recovered by global analysis of progressively quenched samples. Compared with oxytocin, vasopressin displayed a longer correlation time for overall rotational diffusion and a higher amplitude for picosecond segmented motions of its tyrosyl residue. All the data are consistent with a more extended and flexible solution structure for vasopressin than for oxytocin.Dedicated to Professor Alfons Kawski on the occasion of his 65th birthday.     

Picosescond fluorescence lifetime standards for frequency- and time-domain fluorescence

We characterized a series of dimethylamino-stilbene derivatives as standards for time-domain and frequency-domain lifetime measurements. The substances have reasonable quantum yields, are soluble in solvents available with a high purity, and do not show significant sensitivity to oxygen quenching. All the fluorophores displayed single exponential intensity decays, as characterized by frequency-domain measurements to 10 GHz. The decay times vary from 880 to 57 ps, depending on structure, solvent, and temperature, which is a useful range for modern picosecond time-domain or gigahertz frequency-domain instruments. These fluorophores may be used either to test an instrument or as reference compounds to eliminate color effects. We also characterized two-fluorophore mixtures, with the decay times spaced twofold (150 and 300 ps), with varying proportions. These mixtures are useful for testing the resolution of other time- and frequency-domain instrumentation. The excitation wavelength ranges from 260 to 430 nm, and the emission from 350 to 550 nm. The decay times are independent of the excitation and emission wavelengths.     

Fluorescence intensity and anisotropy decay of the 4′,6-diamidino-2-phenylindole-DNA complex resulting from one-photon and two-photon excitation

We examined the emission spectra, intensity decays, and anisotropy decays of the DNA-4,6-diamidino-2-phenylindole (DAPI) complex resulting from one- and two-photon excitation of fluorescence. Similar lifetimes and correlation times were recovered from the frequency-domain data. However, the initial anisotropies of DAPI for one- and two-photon excitation revealed different angles between the absorption and the emission oscillators, 17.8 and 23.8°, respectively. This suggests the presence of two overlapping transitions in DAPI with different one- and two-photon cross sections for absorption.     

Fluorescence intensity and anisotropy decays of the intrinsic tryptophan emission of hemoglobin measured with a 10-GHz fluorometer using front-face geometry on a free liquid surface

We measured the intensity and anisotropy decays of the intrinsic tryptophan emission from hemoglobin solutions obtained using a 10-GHz frequency-domain fluorometer and a specially designed cuvette which allows front-face excitation on a free liquid surface. The cuvette eliminates reflections and stray emissions, which become significant for low-intensity fluorescence such as in hemoglobin. Three lifetimes are detectable in the subnanosecond range. The average lifetime of hemoglobin emission is ligand dependent. The measured values of average lifetimes are 91, 174, and 184 ps for deoxy-, oxy-, and carboxyhemoglobin, respectively. Fluorescence anisotropy decays of oxy-, deoxy-, and carbonmonoxyhemoglobin can be fitted with up to three correlation times. When three components are used, the floating initial anisotropyr _o is, in each case, higher than the steady-state anisotropy of tryptophan in vitrified solution. For deoxy hemoglobin it is close to 0.4. The data are consistent with an initial loss of anisotropy from 0.4 to about 0.3 occurring in the first 2 ps.     

Two photon-induced fluorescence intensity and anisotropy decays of diphenylhexatriene in solvents and lipid bilayers

We measured the fluorescence intensity and anisotropy decays of 1,6-diphenyl-1,3,5-hexatriene (DPH)-labeled membranes resulting from simultaneous two-photon excitation of fluorescence. Comparison of these two-photon data with the more usual one-photon measurements revealed that DPH displayed identical intensity decays, anisotropy decays, and order parameters for one- and two-photon excitation. While the anisotropy data are numerically distinct, they can be compared by use of the factor 10/7, which accounts for the two-photon versus one-photon photoselection. The increased time 0 anisotropy of DPH can result in increased resolution of complex anisotropy decays. Global analysis of the one- and two-photon data reveals consistency with a single apparent angle between the absorption and the emission oscillators. The global anisotropy analysis also suggests that, except for the photoselection factor, the anisotropy decays are the same for one-and two-photon excitation. This ideal behavior of DPH as a two-photon absorber, and its high two-photon cross section, makes DPH a potential probe for confocal two-photon microscopy and other systems where it is advantageous to use long-wavelength (680- to 760-nm) excitation.     

l-Dityrosine: A time-resolved fluorescence investigation

We have investigated the time-resolved fluorescence properties ofll-dityrosine in aqueous solution. Typically, three exponential components were needed to fit the fluorescence pattern adequately, with pure decay terms for the low-intensity, high-energy state (_em = nm) but with a pronounced subnanosecond rise phase for the predominant red-edge fluorescence (_em > 380). Dual fluorescence behavior is indicative of an intramolecular precursorsuccessor pair, i.e., a consecutive intramolecular excited-state reaction. We suggest that this reaction is a torsional motion of the (deprotonated) monoanion along the biphenolic bond. Analysis of the fluorescence anisotropy decay of dityrosine yielded two rotational correlation times, the longer of which is associated with a negative preexponential term. The increase with time in the horizontally polarized component of the intensity decay is interpreted as the result of an electronic rearrangement in the excited state when the successor form of dityrosine is generated. Lifetime distributions of experimental data were probed by an unbiased exponential series method which uses a Tikhonov-type regularization function. The procedure revealed three well-separated groups of lifetimes, the short-lived ensemble forming a formally negative distribution. A photophysical model is introduced which interprets the biexponential decay of dityrosine in terms of overlapping emission signals from the precursor and the successor molecule.     

Dynamics of tRNAtyr Probed with Long-Lifetime Metal-Ligand Complexes

The metal-ligand complexes, [Ru(bpy)₂(dppz)]²⁺ (bpy = 2,2??-bipyridine, dppz = dipyrido[3,2-a:2??,3??-c]phenazine) (RuBD) and [Ru(phen)₂(dppz)]²⁺ (phen = 1,10-phenanthroline) (RuPD), display favorable photophysical properties including long lifetime, polarized emission, and very little background fluorescence. To check if RuBD and RuPD reflect the overall rotational mobility of small nucleic acid, we measured the intensity and anisotropy decays of RuBD and RuPD when intercalated into tRNA^tyr using pBC SK(+) phagemid as a control. We used frequency-domain fluorometry with a blue light-emitting diode (LED) as the modulated light source. We observed shorter lifetimes for tRNA^tyr than those for the pBC SK(+) phagemid for both probes, however, RuPD showed much larger decrease in the mean lifetime values (64%). The slow rotational correlation time of RuBD (31.3 ns) and the fast rotational correlation time of RuPD (26.0 ns) reflected the overall rotational mobility of tRNA^tyr. In addition, the steady-state anisotropy and time-resolved anisotropy decay data showed a clear difference between tRNA^tyr and pBC SK(+) phagemid. This suggests the possibility of a homogeneous assay for identifying target nucleic acids and/or nucleic acid binding proteins.     

A General Method for Constrained Analysis of Fluorescence Anisotropy Decay: Application of the Steady-State Anisotropy

Time-resolved fluorescence anisotropy is an invaluable method for investigating the internal and rotational dynamics of biomolecules. The range of rotational motions detectable by anisotropy decay is limited by the fluorescence lifetime; typically, a depolarizing motion may be resolved if the associated correlation time is between 0.1 and 10 times the intensity decay lifetime. To extend that range and to improve the recovery of anisotropy decay parameters, a general analytical method has been developed. This procedure utilizes a modification of Lagrange multiplier methods to constrain the values of the iterated kinetic parameters during nonlinear least-squares analysis of anisotropy decay data. The form of the constraint equation is derived from the classic relationship between the decay parameters and the steady-state anisotropy, which can be simply and accurately measured. Application of the constraint to analyses of synthetic data sets increased the accuracy of recovery by decreasing the uncertainty in the iterated parameters. The constraint also enabled the accurate recovery of correlation times that were a factor of 30 greater than the fluorescence lifetime, although it did not improve recovery of correlation times that were much shorter than the lifetime. Using this technique, it should now be possible to characterize the dynamics of larger macromolecules and assemblies than those that can currently be studied by fluorescence anisotropy decay.     

Perturbation of Conformational Dynamics of ASCUT-1 from Ascaris lumbricoides by Temperature and Sodium Dodecyl Sulfate

ASCUT-1 is a protein found in cuticlin, the insoluble residue of the cuticles of the nematode Ascaris lumbricoides. It contains the CUT-1-like domain which is shared by members of a novel family of components of extracellular matrices. The monomeric form of ASCUT-1 contains a single tryptophan residue. An understanding of the structure-function relationship of the protein under different chemical-physical conditions is of fundamental importance for an understanding of its structure and function in cuticles. In this paper we report the effect of the temperature and sodium dodecyl sulfate on the structural stability of this protein. The structure of the protein was studied in the temperature range 25–85°C in the absence and in the presence of sodium dodecyl sulfate by frequency-domain measurements of the intrinsic fluorescence intensity and anisotropy decays. The time-resolved fluorescence data in the absence of SDS indicated that the tryptophanyl emission decays were well described by a bimodal lifetime distribution, and that the temperature increases resulted in the sharpening and in the shortening of the tryptophanyl lifetime distribution. In the presence of SDS an unimodal fluorescence lifetime distribution as well as a marked decrease in the anisotropy decay values were observed.     

Three-photon-induced fluorescence of diphenylhexatriene in solvents and lipid bilayers

We observed the emission of l,6-diphenyl-l,3,5-hexatriene (DPH) when excited with the fundamental output of a fs Ti:sapphire laser at 860 nm. The emission spectra of DPH were identical to that observed for one-photon excitation at 287 nm. The dependence of the DPH emission intensity on laser power was cubic, indicating three-photon excitation of DPH at 860 nm. At a shorter wavelength of 810 nm, the dependence on laser power was quadratic, indicating a two-photon process. At an intermediate wavelength of 830 nm the mode of excitation was a mixture of two- and three-photon excitation. At 830 nm the anisotropy is no longer a molecular parameter, and the mode of excitation and anisotropy of DPH depends on laser power. Frequency-domain anisotropy decays of DPH in triacetin revealed the same rotational correlation times for two- and three-photon excitation. However, the time 0 anisotropy of DPH was larger for three-photon excitation than for two-photon excitation. Steady-state anisotropy data for DPH-labeled membranes revealed the same transition temperature for one- and three-photon excitation. These anisotropy data indicate that membrane heating was not significant with three-photon excitation and that three-photon excitation may thus be of practical usefulness in fluorescence spectroscopy and microscopy of membranes.     

Effects of light quenching on the emission spectra and intensity decays of fluorophore mixtures

We examined a series of fluorophore mixtures to determine the wavelength selectivity of light quenching and the effects of light quenching on the emission spectra and intensity decays. Light quenching can be accomplished using a single excitation pulse train and quenching wavelength (one-beam) or with longer-wavelength quenching pulses time-delayed relative to the excitation pulses (two-beam). Both one-beam and two-beam light quenching were found to alter the intensity decays of the mixtures. The frequency-domain intensity decay data were analyzed to reveal the fractional intensity of each fluorophore in the mixture and the effects of light quenching on the fractional contribution of each fluorophore to the total intensity. Fluorophores were selected to provide a range of decay times and emission wavelengths. The extent of quenching in the mixtures was dependent on which fluorophore had the higher radiative decay rate and emission intensity at the quenching wavelength. A general theory is presented which describes the intensity decays in terms of the extent of quenching of each fluorophore and the time delay between excitation and quenching pulses. The effects of light quenching on the fractional intensities of each fluorophore in the mixture, recovered from the intensity decay analysis, were found to be in quantitative agreement with that predicted from steady-state measurements of light quenching and from the spectral properties of the fluorophores. The data on light quenching of mixtures demonstrate that light quenching may be used for selective quenching of fluorophores and thus of potential value for studies of multichromophoric systems.     

Dynamics of Bacteriophage R17 Probed with a Long-Lifetime Ru(II) Metal-Ligand Complex

The metal-ligand complex, [Ru(2,2′-bipyridine)₂(4,4′-dicarboxy-2,2′-bipyridine)]²⁺ (RuBDc), was used as a spectroscopic probe for studying macromolecular dynamics. RuBDc is a very photostable probe that possesses favorable photophysical properties including long lifetime, high quantum yield, large Stokes’ shift, and highly polarized emission. To further show the usefulness of this luminophore for probing macromolecular dynamics, we examined the intensity and anisotropy decays of RuBDc when conjugated to R17 bacteriophage using frequency-domain fluorometry with a blue light-emitting diode (LED) as the modulated light source. The intensity decays were best fit by a sum of two exponentials, and we obtained a longer mean lifetime at 4 °C (<τ> = 491.8 ns) as compared to that at 25 °C (<τ> = 435.1 ns). The anisotropy decay data showed a single rotational correlation time, which is typical for a spherical molecule, and the results showed a longer rotational correlation time at 4 °C (2,574.9 ns) than at 25 °C (2,070.1 ns). The use of RuBDc enabled us to measure the rotational correlation time up to several microseconds. These results indicate that RuBDc has significant potential for studying hydrodynamics of biological macromolecules.     

Effects of Refractive Index and Viscosity on Fluorescence and Anisotropy Decays of Enhanced Cyan and Yellow Fluorescent Proteins

The fluorescence lifetime strongly depends on the immediate environment of the fluorophore. Time-resolved fluorescence measurements of the enhanced forms of ECFP and EYFP in water–glycerol mixtures were performed to quantify the effects of the refractive index and viscosity on the fluorescence lifetimes of these proteins. The experimental data show for ECFP and EYFP two fluorescence lifetime components: one short lifetime of about 1 ns and a longer lifetime of about 3.7 ns of ECFP and for EYFP 3.4. The fluorescence of ECFP is very heterogeneous, which can be explained by the presence of two populations: a conformation (67% present) where the fluorophore is less quenched than in the other conformation (33% present). The fluorescence decay of EYFP is much more homogeneous and the amplitude of the short fluorescence lifetime is about 5%. The fluorescence anisotropy decays show that the rotational correlation time of both proteins scales with increasing viscosity of the solvent similarly as shown earlier for GFP. The rotational correlation times are identical for ECFP and EYFP, which can be expected since both proteins have the same shape and size. The only difference observed is the slightly lower initial anisotropy for ECFP as compared to the one of EYFP.     

Long-lifetime metal-ligand complexes as luminescent probes for DNA

We examined the intensity and anisotropy decays of DNA labeled with two ruthenium metalligand complexes, [Ru(bpy)₂(dppz)]²⁺ and [Ru(phe)₂(dppz)]²⁺. Both complexes display high emission anisotropies in the absence of rotational diffusion, making them suitable probes for rotational motions. When bound to DNA, these complexes display decay times as long as 294 ns, providing long-lived probes of DNA dynamics. The decay times of both complexes were rather insensitive to dissolved oxygen. We examined anisotropy decays of these complexes bound to B-form DNA. The anisotropy decays revealed correlation times near 10, 50, and several hundred nanoseconds, suggesting that these probes are sensitive to a wide range of DNA motions. The use of metalligand complexes should allow resolution of both the torsional and bending motions of DNA, the latter of which has been mostly inaccessible using shorter-lived fluorescent probes bound to DNA. Dedicated to Professor Robert F. Steiner upon his retirement     

Fluorescence of horse liver alcohol dehydrogenase using one- and two-photon excitation

We examined the steady-state and time-resolved emission of liver alcohol dehydrogenase resulting from one-photon and two-photon excitation. Previous studies with one-photon excitation revealed that the two nonidentical tryptophan residues display different emission spectra and decay times. The use of two-photon excitation resulted in similar emission spectra, multiexponential intensity decays, time-resolved emission spectra, and anisotropy decays as was observed for one-photon excitation. These results suggest that both nonidentical tryptophan residues are excited to a similar extent for one- and two-photon excitation. However, the limiting anisotropy (r ₀) with two-photon excitation from 585 to 610 nm is below 0.1 and appears distinct from that observed previously forN-acetyl-l-tryptophanamide.Abbreviations LADH liver alcohol dehydrogenase - -NAD⁺ -nicotinamide adenine dinucleotide - OPE one-photon excitation - OPIF one-photon induced fluorescence - TPE two-photon excitation - TCSPC time-correlated single photon counting - TPIF two-photon induced fluorescence     

Time-resolved fluorescence studies of ribonuclease T1 in reversed micelles

Time-resolved fluorescence intensity and anisotropy decay data were obtained for ribonuclease T₁ entrapped in bis(2-ethylhexyl) sodium sulfosuccinate/heptane reverse micelles, as a function of the size of the inner water pool at neutral pH. Data have been presented previously to show that this protein retains its native structure and undergoes reversible thermal unfolding in these reverse micelles (Shastry and Eftink,Biochemistry 36, in press). The fluorescence decay of entrapped protein is similar to that for the protein in buffer. The rotational correlation time of entrapped ribonuclease T₁ is found to be longer than that in buffer; this rotational correlation time decreases with increasing size of the water pool but is still over twice the value for the protein in buffer for the largest size of water pool investigated, indicating an increased microviscosity within the reverse micelle. Thermal unfolding of the protein results in a significant decrease in the rotational correlation time of the entrapped proteins, consistent with the protein being unfolded but not interacting with the inner surfactant wall of the reverse micelle.Nomenclature _i Amplitude of component i associated with fluorescence decay _i - _i Fractional intensity associated with fluorescence decay time _i - Rotational correlation time g_i Amplitude of component i associated with anisotropy decay _i - _max Fluorescence emission maximum - r_o Fundamental anisotropy of an immobilized fluorophore - _i Fluorescence lifetime of component i - W_o Ratio of water molecules per detergent molecules in a reverse micelle     

Fluorescence and Rotational Dynamics of Dityrosine

We have examined the lifetimes and rotational correlation times of dityrosine emission by time-correlated single-photon counting. We first noticed dityrosine fluorescence in samples of tyrosine and tyrosine dipeptides by its characteristic red-shifted emission at 400 to 430 nm. The longer rotational correlation time relative to tyrosine proved that this fluorescence emanated from a distinct species. Comparison with the fluorescence properties of synthesized dityrosine established the identity of the emitting species. Fluorescence intensity decays of dityrosine are generally characterized by two decay components, one with a lifetime in the range of 150 to 800 ps and another between 2.5 and 4.5 ns. We found no evidence for an excited-state reaction, since a rising phase (negative-amplitude component) was not observed. In the pH range from 4 to 10, two ground-state species exist in equilibrium with pK _a 7. Both species exhibit two fluorescence decays. The average fluorescence lifetime increases gradually with pH over the pH range from 4 to 10 and decreases at pH 2. Anisotropy decays were measured for dityrosine and the alanine–dityrosine–alanine and leucine–dityrosine–leucine dipeptides. The rotational correlation times of dityrosine and dityrosine dipeptides increase linearly with van der Waals volumes. The slope indicates a stronger solute–solvent interaction than predicted with stick boundary conditions. It is suggested that these interactions result from the presence of two zwitterionic pairs.     

Light quenching of tetraphenylbutadiene fluorescence observed during two-photon excitation

We observed the steady-state and time-resolved emission of tetraphenylbutadiene (TPB) whea excited by simultaneous absorption of two photons (514 to 610 nm). The intensity initially increased quadratically with laser power, as expected for a two-photon process. At higher laser powers the intensity increases in TPB were subquadratic. The intensity and anisotropy decay times of TPB were unchanged under the locally intense illumination. Importantly, the time zero anisotropy of TPB was decreased under conditions where the intensity was subquadratic. Furthermore, the subquadratic dependence on incident power was not observed for two-photon excitation of 2,5-diphenyloxazole (PPO), for which the incident wavelength does not overlap with the emission spectrum. These results are consistent with stimulated emission (light quenching) of TPB at high laser intensities. The phenomenon of light quenching may be important for other fluorophores used in biochemical research, particularly for the high local intensities used for two-photon excitation.     

Dynamics ofLens culinaris agglutinin studied by red-edge excitation spectra and anisotropy measurements of 2-p-toluidinylnaphthalene-6-sulfonate (TNS) and of tryptophan residues

The fluorescence of 2-p-toluidinylnaphthalene-6-sulfonate bound toLens culinaris agglutinin and of the Trp residues of the protein was investigated. Red-edge excitation spectra and steady-state anisotropy as a function of temperature indicate that the TNS is bound rigidly. Red-edge excitation spectra, steady-state anisotropy as a function of sucrose and anisotropy decay experiments performed on Trp residues fluorescence prove that the internal fluorophore presents residual motion independent of the global rotation of the protein. Fluorescence anisotropy decay allows to calculate the rotational correlation time (351 ps) of this local motion. Quenching resolved emission anisotropy with iodide gives values equal to 0.257 and 0.112 for the anisotropies of the buried and the surface Trp residues, respectively. This result indicates that the Trp residues present at the surface of the protein have important local motions compared to those embedded in the protein matrix. The results obtained from TNS and Trp residues indicate that the agglutinin has different dynamic domains.