Time Evolution of Local pH Around a Photo-Acid in Water and a Polymer Hydrogel: Time Resolved Fluorescence Spectroscopy of Pyranine

In this work, we propose a new analysis of the time resolved emission spectra of a photo-acid, HA, pyranine (8-hydroxypyrene-1,3,6-trisulphonic acid, HPTS) based on time resolved area normalized emission spectra (TRANES). Presence of an isoemissive point in TRANES confirms the presence of two emissive species (HA and A⁻) inside the system in bulk water and inside a co-polymer hydrogel [F127, (PEO)₁₀₀–(PPO)₇₀–(PEO)₁₀₀]. We show that following electronic excitation, the local pH around HPTS, is much lower than the bulk pH presumably because of ejection of proton from the photo-acid in the excited state. With increase in time, the local pH increases and reaches the bulk value. We further, demonstrate that the excited state pKa of HPTS may be estimated from the emission intensities of HA and A⁻ at long time. The time constant for time evolution of pH is ∼630 ps in water, ∼1300 ps in F127 gel and ∼4700 ps in CTAB micelle. The location and local viscosity sensed by the probe is ascertained using fluorescence correlation spectroscopy (FCS) and fluorescence anisotropy decay. The different values of the local viscosity reported by these two methods are reconciled.     

Existence of a new emitting singlet state of proflavine: femtosecond dynamics of the excited state processes and quantum chemical studies in different solvents

Proflavine (3,6-diaminoacridine) shows fluorescence emission with lifetime, 4.6 ± 0.2 ns, in all the solvents irrespective of the solvent polarity. To understand this unusual photophysical property, investigations were carried out using steady state and time-resolved fluorescence spectroscopy in the pico- and femtosecond time domain. Molecular geometries in the ground and low-lying excited states of proflavine were examined by complete structural optimization using ab initio quantum chemical computations at HF/6-311++G** and CIS/6-311++G** levels. Time dependent density functional theory (TDDFT) calculations were performed to study the excitation energies in the low-lying excited states. The steady state absorption and emission spectral details of proflavine are found to be influenced by solvents. The femtosecond fluorescence decay of the proflavine in all the solvents follows triexponential function with two ultrafast decay components (τ(1) and τ(2)) in addition to the nanosecond component. The ultrafast decay component, τ(1), is attributed to the solvation dynamics of the particular solvent used. The second ultrafast decay component, τ(2), is found to vary from 50 to 215 ps depending upon the solvent. The amplitudes of the ultrafast decay components vary with the wavelength and show time dependent spectral shift in the emission maximum. The observation is interpreted that the time dependent spectral shift is not only due to solvation dynamics but also due to the existence of more than one emitting state of proflavine in the solvent used. Time resolved area normalized emission spectral (TRANES) analysis shows an isoemissive point, indicating the presence of two emitting states in homogeneous solution. Detailed femtosecond fluorescence decay analysis allows us to isolate the two independent emitting components of the close lying singlet states. The CIS and TDDFT calculations also support the existence of the close lying emitting states. The near constant lifetime observed for proflavine in different solvents is suggested to be due to the similar dipole moments of the ground and the evolved emitting singlet state of the dye from the Franck-Condon excited state.     

Solvation dynamics of DCM in a polypeptide-surfactant aggregate: gelatin-sodium dodecyl sulfate

Solvation dynamics of 4-(dicyanomethylidene)-2-[p-(dimethylamino)styryl]-6-methyl-4H-pyran (DCM) is studied in a polypeptide-surfactant aggregate consisting of gelatin and sodium dodecyl sulfate (SDS) in potassium dihydrogen phosphate (KP) buffer. The average solvation time (tauS) in gelatin-SDS aggregate at 45 degrees C is found to be 1780 ps, which is about 13 times slower than that in 15 mM SDS in KP buffer at the same temperature. The fluorescence anisotropy decay in gelatin-SDS aggregate is also different from that in SDS micelles in KP buffer. DCM displays negligible emission in the presence of gelatin in aqueous solution. Thus the solvation dynamics in the presence of gelatin and SDS is exclusively due to the probe (DCM) molecules at the gelatin-micelle interface. The slow solvation dynamics is ascribed to the restrictions imposed on the water molecules trapped between the polypeptide chain and micellar aggregates. The critical association concentration (cac) of SDS for gelatin is determined to be 0.5 +/- 0.1 mM.     

Competition between energy and proton transfer in ultrafast excited-state dynamics of an oligomeric fluorescent protein red Kaede

We investigated femtosecond and picosecond time-resolved fluorescence dynamics of a tetrameric fluorescent protein Kaede with a red chromophore (red Kaede) to examine a relationship between the excited-state dynamics and a quaternary structure of the fluorescent protein. Red Kaede was obtained by photoconversion from green Kaede that was cloned from a stony coral Trachyphyllia geoffroyi. In common with other typical fluorescent proteins, a chromophore of red Kaede has two protonation states, the neutral and the anionic forms in equilibrium. Time-resolved fluorescence measurements clarified that excitation of the neutral form gives the anionic excited state with a time constant of 13 ps at pH 7.5. This conversion process was attributed to fluorescence resonance energy transfer (FRET) from the photoexcited neutral form to the ground-state anionic form that is located in an adjacent subunit in the tetramer. The time-resolved fluorescence data measured at different pH revealed that excited-state proton transfer (ESPT) also occurs with a time constant of 300 ps and hence that the FRET and ESPT take place simultaneously in the fluorescent protein as competing processes. The ESPT rate in red Kaede was significantly slower than the rate in Aequorea GFP, which highly likely arises from the different hydrogen bond network around the chromophore.     

Rotational reorientation dynamics of oxazine 750 in polar solvents

The rotational reorientation dynamics of oxazine 750 (OX750) in the first (with pump pulse at 660 nm) and a higher excited state (with pump pulse at 400 nm) in different polar solvents have been investigated using femtosecond time-resolved stimulated emission pumping fluorescence depletion (FS TR SEP FD) spectroscopy. In both excited states, three different anisotropy decay laws have been observed for OX750 in different solvents. Only in acetone and formamide could the anisotropy decays of OX750 be described by single-exponential functions, whereas the anisotropy decays have been found to exhibit biexponential behavior in other solvents. The slower anisotropy decay observed in all of the solvents has been assigned to the overall rotational relaxation of OX750 molecules, and a quantitative analysis of this time constant has been performed using the Stokes-Einstein-Debye hydrodynamic theory and the extended charge distribution model developed by Alavi and Waldeck. In both methanol and ethanol, a faster anisotropy decay on the order of picoseconds and a slower anisotropy decay on the hundreds of picoseconds time scale are observed. The most likely explanation for the faster anisotropy involves the rotation of the transition dipole moment in the excited state of OX750 resulting from the electron transfer (ET) reaction taking place from the alcoholic solvents to the OX750 chromophore. As a possible explanation, the wobbling-in-the-cone model has been used to analyze the biexponential anisotropy decays of OX750 in dimethylformamide (DMF) and dimethyl sulfoxide (DMSO). The observed faster anisotropy decays on the hundreds of femtoseconds time scale in DMF and DMSO are ascribed to the wobbling-in-the-cone motion of the ethyl group of OX750, which is sensitive to the strength of the hydrogen bond formed between the solvent and the protonation site of OX750.     

Ultrafast surface solvation dynamics and functionality of an enzyme alpha-chymotrypsin upon interfacial binding to a cationic micelle

In this contribution we report studies on enzymatic activity of alpha-chymotrypsin (CHT) upon complexation with cationic cetyltrimethylammonium bromide (CTAB) micelle. With picosecond time resolution, we examined solvation dynamics at the interface of CHT-micelle complex, and rigidity of the binding. We have used 5-(dimethyl amino) naphthalene-1-sulfonyl chloride (dansyl chloride; DC) that is covalently attached to the enzyme at the surface sites. The solvation processes at the surface of CHT in buffer solution are found to be mostly in the sub-50 ps time scale. However, at the interface the solvation correlation function decays with time constant 150 ps (65%) and 500 ps (35%), which is significantly different from those found at the enzyme and micellar surfaces. The binding structure of the enzyme-micelle complex was examined by local orientational motion of the probe DC and compared with the case without micelle. The orientational dynamics of the probe DC in the complex reveals a structural perturbation at the surface sites of CHT upon complexation, consistent with other reported structural studies. We also found possible entanglement of charge transfer dynamics of the probe DC on the measured solvation processes by using time-resolved area normalized emission spectroscopic technique. The interfacial solvation process and complex rigidity elucidate the strong recognition mechanism between CHT and the micelle, which is important to understand the biological function of CHT upon complexation with the micelle.     

Photophysical properties of DASPMI as revealed by spectrally resolved fluorescence decays

Photophysical properties of 2-(4-(dimethylamino)styryl)-1-methylpyridinium iodide (DASPMI) in various solvents were investigated using time- and space-correlated single photon counting. DASPMI is known to selectively stain mitochondria in living cells.1,2 The uptake and fluorescence intensity of DASPMI in mitochondria is a dynamic measure of membrane potential. Hence, an endeavor has been made to elucidate the mechanism of DASPMI fluorescence by obtaining spectrally resolved fluorescence decays in different solvents. A biexponential decay model was sufficient to globally describe the wavelength-dependent fluorescence in ethanol and chloroform. While in glycerol, a three-exponential decay model was necessary for global analysis. In the polar low-viscous solvent water, a monoexponential decay model fitted the decay data. The sensitivity of DASPMI to solvent viscosity was analyzed using various proportions of glycerol-ethanol mixtures. The lifetimes were found to increase with increasing solvent viscosity. The negative amplitudes of the short lifetime component found in chloroform and glycerol at the longer wavelengths validated the formation of new excited-state species from the initially excited state. Time-resolved emission spectra in chloroform and glycerol showed a biphasic increase of spectral width and emission maxima. The spectral width had an initial fast increase within 150 ps and a near constant thereafter. A three-state model of generalized scheme, on the basis of successive formation of locally excited state (LE), intramolecular charge transfer state (ICT), and twisted intramolecular charge transfer (TICT) state, has been proposed to explain the excited-state kinetics. The presumed role of solvation dynamics of ICT and TICT states leading to the asymmetrical broadening and structureless fluorescence has been substantiated by the decomposition of time-resolved emission spectra in chloroform, glycerol, and ethanol/glycerol mixtures.     

Intrachain energy migration to weak charge-transfer state in polyfluorene end-capped with naphthalimide derivative

Polyfluorene end-capped with N-(2-benzothiazole)-1,8-naphthalimide (PF-BNI) is a highly fluorescent material with fluorescence emission modulated by solvent polarity. Its low energy excited state is assigned as a mixed configuration state between the singlet S(1) of the fluorene backbone (F) with the charge transfer (CT) of the end group BNI. The triexponential fluorescence decays of PF-BNI were associated with fast energy migration to form an intrachain charge-transfer (ICCT) state, polyfluorene backbone decay, and ICCT deactivation. Time-resolved fluorescence anisotropy exhibited biexponential relaxation with a fast component of 12-16 ps in addition to a slow one in the range 0.8-1.4 ns depending on the solvent, showing that depolarization occurs from two different processes: energy migration to form the ICCT state and slow rotational diffusion motion of end segments at a longer time. Results from femtosecond transient absorption measurements agreed with anisotropy decay and showed a decay component of about 16 ps at 605 nm in PF-BNI ascribed to the conversion of S(1) to the ICCT excited state. From the ratio of asymptotic and initial amplitudes of the transient absorption measurement, the efficiency of intrachain ICCT formation is estimated in 0.5, which means that, on average, half of the excited state formed in a BNI-(F)(n)-BNI chain with n = 32 is converted to its low energy intrachain charge-transfer (ICCT) state.     

TRANES analysis of the fluorescence of nile red in organized molecular assemblies confirms emission from two species

Time-resolved area normalized emission spectroscopy (TRANES) is a new method for the analysis of fluorescence of dyes in complex chemical and biological systems (A S R Koti, M M G Krishna and N Periasamy, 2001,J. Phys. Chem. 105, 1767). The model-free method extends the power of time-resolved emission spectroscopy (TRES) analysis and removes the ambiguity in the interpretation when the emission spectrum is time-dependent. Observation of an isoemissive point in TRANES analysis of fluorescence is an unambiguous indication for the presence of two emissive species in the sample. The isoemissive point occurs at a wavelength where the ratio of the radiative rates of the two species is equal to the ratio of their total radiative rates. The polarity-sensitive nile red dye shows time-dependent emission spectra in the organized bilayer assemblies of TX micelle and bilayer egg-phosphotidylcholine (egg-PC) membrane. Time-dependent spectra in complex systems support many important models (solvation model and heterogeneity in the ground and/or excited state). TRANES analysis shows that the fluorescence emission of nile red in TX micelle and egg-PC membrane is due to two emissive species solubilized in different sites.     

Different sources of 4-aminophthalimide solvation dynamics retardation inside micellar systems

The solvation dynamics of 4-aminophthalimide (4-AP) in two micellar systems (cetyltrimethylammonium bromide (M-CTAB) and Triton X-100 (M-TX-100)) has been studied. The results presented are in agreement with our earlier findings in sodium dodecyl sulfate (M-SDS) micelle (J. Phys. Chem. B 107 (2003) 13,986, J. Phys. Chem. B 109 (2005) 9422). They have confirmed that the main reason for the observed shape and position of the time changes in 4-AP time-resolved emission spectra (TRES) is the process of establishing a new equilibrium between two emissive species present in micellar systems, the excited 4-AP in the intramolecular charge transfer state (S(1)-ICT) and the exciplex formed between 4-AP in the S1-ICT state and water molecules dissolved inside micelles. In M-TX-100 and in M-CTAB this process has been found to be slower than in the earlier studied M-SDS. The presence of two emitting species has been concluded on the basis of observation of the isoemissive point in the time-resolved area-normalized emission spectra (TRANES) of 4-AP in micellar systems studied. It has been shown that the distance between the 4-AP molecule and the water molecules present inside the micelles can be one of the parameters responsible for the long-time duration of the exciplex formation and solvation process in the micelles.     

Solvation dynamics of DCM in a DPPC vesicle entrapped in a sodium silicate derived sol-gel matrix

Solvation dynamics of 4-(dicyanomethylene)-2-methyl-6(p-dimethylaminostyryl) 4H-pyran (DCM) has been studied in a dipalmitoyl-phosphatidylcholine (DPPC) vesicle entrapped in a sodium silicate derived sol-gel glass. Solvation dynamics in DPPC in a sol-gel glass is described by two components of 350 +/- 50 ps (50%) and 2300 +/- 200 ps (50%) with a total dynamic Stokes shift of 1300 cm(-1). The fast component (350 ps) is similar to the fast component in a DPPC vesicle in bulk water (320 +/- 50 ps). This component may be ascribed to the dynamics of the water molecules inside the water pool of the vesicle. However, the slow component (2300 +/- 200 ps) is about 2.5 times slower compared to the slow component of solvation dynamics of DCM in a DPPC vesicle in bulk solvent (900 +/- 100 ps). The anisotropy decay of DCM in a DPPC vesicle both in sol-gel glass and in bulk water exhibits a very fast initial decay with a large residual anisotropy, which does not decay in approximately 10 ns. The time scale of anisotropy decay is very different from that of solvation dynamics.     

Temperature dependence of solvation dynamics and anisotropy decay in a protein: ANS in bovine serum albumin

Temperature dependence of solvation dynamics and fluorescence anisotropy decay of 8-anilino-1-naphthalenesulfonate (ANS) bound to a protein, bovine serum albumin (BSA), are studied. Solvation dynamics of ANS bound to BSA displays a component (300 ps) which is independent of temperature in the range of 278-318 K and a long component which decreases from 5800 ps at 278 K to 3600 ps at 318 K. The temperature independent part is ascribed to a dynamic exchange of bound to free water with a low barrier. The temperature variation of the long component of solvation dynamics corresponds to an activation energy of 2.1 kcal mol(-1). The activation energy is ascribed to local segmental motion of the protein along with the associated water molecules and polar residues. The time scale of solvation dynamics is found to be very different from the time scale of anisotropy decay. The anisotropy decays are analyzed in terms of the wobbling motion of the probe (ANS) and the overall tumbling of the protein.     

Femtosecond excited-state dynamics of 4-nitrophenyl pyrrolidinemethanol: evidence of twisted intramolecular charge transfer and intersystem crossing involving the nitro group

Ultrafast excited-state relaxation dynamics of a nonlinear optical (NLO) dye, (S)-(-)-1-(4-nitrophenyl)-2-pyrrolidinemethanol (NPP), was carried out under the regime of femtosecond fluorescence up-conversion measurements in augmentation with quantum chemical calculations. The primary concern was to trace the relaxation pathways which guide the depletion of the first singlet excited state upon photoexcitation, in such a way that it is virtually nonfluorescent. Ground- and excited-state (singlet and triplet) potential energy surfaces were calculated as a function of the -NO(2) torsional coordinate, which revealed the perpendicular orientation of -NO(2) in the excited state relative to the planar ground-state conformation. The fluorescence transients in the femtosecond regime show biexponential decay behavior. The first time component of a few hundred femtoseconds was ascribed to the ultrafast twisted intramolecular charge transfer (TICT). The occurrence of charge transfer (CT) is substantiated by the large dipole moment change during excitation. The construction of intensity- and area-normalized time-resolved emission spectra (TRES and TRANES) of NPP in acetonitrile exhibited a two-state emission on behalf of decay of the locally excited (LE) state and rise of the CT state with a Stokes shift of 2000 cm(-1) over a time scale of 1 ps. The second time component of a few picoseconds is attributed to the intersystem crossing (isc). In highly polar solvents both the processes occur on a much faster time scale compared to that in nonpolar solvents, credited to the differential stability of energy states in different polarity solvents. The shape of frontier molecular orbitals in the excited state dictates the shift of electron density from the phenyl ring to the -NO(2) group and is attributed to the charge-transfer process taking place in the molecule. The viscosity dependence of relaxation dynamics augments the proposition of considering the -NO(2) group torsional motion as the main excited-state relaxation coordinate.     

Picosecond multidimensional fluorescence spectroscopy: a tool to measure real-time protein dynamics during function

Advanced multidimensional time-correlated single photon counting (mdTCSPC) and picosecond time-resolved fluorescence in combination with site-directed fluorescence labeling are valuable tools to study the properties of membrane protein surface segments on the pico- to nanoseconds time scale. Time-resolved fluorescence anisotropy changes of protein bound fluorescent probes reveal changes in protein dynamics and steric restriction. In addition, the change in fluorescence lifetime and intensity of the covalently bound fluorescent dye is indicative of environmental changes at the protein surface. In this study, we have measured the changes in fluorescence lifetime traces of the fluorescent dye fluorescein covalently bound to the first cytoplasmic loop of bacteriorhodopsin (bR) after light activation of protein function. The fluorescence is excited by a picosecond laser pulse. The retinylidene chromophore of bR is light-activated by a 10 ns laser pulse, which in turn triggers recording of a sequence of fluorescence lifetime traces in the mdTCSPC-module. The fluorescence decay changes upon protein function occur predominantly in the 100 ps time range. The kinetics of these changes shows two transitions between three intermediate states in the second part of the bR photocycle. Correlation with photocycle kinetics allows for the determination of reaction intermediates at the proteins surface which are coupled to changes in the retinal binding pocket.     

Fluorescence anisotropy decay and solvation dynamics in a nanocavity: coumarin 153 in methyl beta-cyclodextrins

Fluorescence anisotropy decay and solvation dynamics of coumarin 153 (C153) are studied in dimethyl beta-cyclodextrin (DIMEB) and trimethyl beta-cyclodextrin (TRIMEB) nanocavity in water. C153 binds to DIMEB and TRIMEB to form both 1:1 and 1:2 (C153:cyclodextrin) complexes. The anisotropy decays of C153 in DIMEB and TRIMEB are found to be biexponential. The fast component of anisotropy decay (approximately 1000 ps) is attributed to the 1:1 complex and the slower one (approximately 2500 ps) to the 1:2 complex. From the components of the anisotropy decay, the length of the 1:1 and 1:2 complexes are estimated. Solvation dynamics of C153 in DIMEB exhibits a very fast (2.4 ps) component (41%) and two slower components of 50 ps (29%) and 1450 ps (30%). Solvation dynamics in TRIMEB is described by three slow components of 10.3 ps (24%), 240 ps (45%), and 2450 ps (31%). Possible origins of the ultraslow components are discussed.     

Effect of nanocavity confinement on the rotational relaxation dynamics: 3-acetyl-4-oxo-6,7-dihydro-12H indolo-[2,3-a] quinolizine in micelles

In continuation of our recent study on the steady state photophysics of a biologically active beta-carboline derivative, 3-acetyl-4-oxo-6,7-dihydro-12H indolo-[2,3-a] quinolizine (AODIQ), in the present article we have investigated the effect of nanocavity confinement on the excited state dynamics and rotational relaxation of the probe using picosecond time resolved fluorescence and fluorescence anisotropy techniques. The polarity dependent intramolecular charge transfer process is responsible for the remarkable sensitivity of this biological fluorophore in micellar environments. The fluorescence anisotropy decay of AODIQ incorporated inside the micelle is biexponential. The rotational motion of the probe was interpreted on the basis of a two step model consisting of a fast restricted rotation of the probe and a slow lateral diffusion of the probe in the micelle; both coupled to the overall rotation of the micelle. Experimental results reveal that micellar environment causes significant retardation of both the wobbling as well as the translational motion of the probe.     

Time-resolved emission of flavin adenine dinucleotide in water and water-methanol mixtures

Time-resolved fluorescence decay of flavin adenine dinucleotide (FAD) was studied at room temperature in water and water-methanol mixtures by a fluorescence upconversion technique. The observations were focused on the most initial decay phase (200 ps), before the residual fluorescence assumes a single exponential decay, typical for an extended conformation of the fluorophore. Within the first few picoseconds, where most of the electron transfer coupled quenching takes place, the emission decay curves could be fitted by a stretched exponent, compatible with the inhomogeneous distance dependent electron transfer model. This implies that the population of the excited FAD molecules exhibits a large number of non-identical states, each with its own separation between the donor (adenine) and acceptor (isoalloxazine) moieties, having its own rate of electron transfer. To evaluate the distribution of the separation between the donor-acceptor pair, we carried out molecular dynamics simulations of closed conformation of the FAD in water and water-methanol mixtures, sampling the structure at 10 fs intervals. The analysis of the dynamics reveals that within the 4 ps time frame, where most of the nonexponential fluorescence relaxation takes place, the relative motion of the donor-acceptor pair is consistent with a one-dimensional Brownian motion, where the diffusion coefficient and the shape of the confining potential well are solvent dependent. The presence of methanol enhances the diffusion constant and widens the width of the potential well. On the basis of these parameters, the relaxation dynamics was accurately reconstructed as an electron transfer reaction in an inhomogeneous system where the reactants are diffusing within the time frame of the observation.     

TIME-RESOLVED SPECTROSCOPIC STUDIES ON PHOTOPHYSICAL PROPERTIES OF BENOXAPROFEN IN MICELLAR SOLUTIONS: AN INTRAMICELLAR FLUORESCENCE QUENCHING

The micellar dependencies of the photophysical properties of benoxaprofen (BXP), a 2-phenyl benzoxazole derivative, have been investigated using fluorescence spectroscopy and laser flash photolysis techniques. The fluorescence of BXP in aqueous solution has been observed to be remarkably quenched upon addition of a surfactant, cetyltrimethyl ammonium bromide (CTAB) or Triton X-100, in contrast to its enhancement in sodium dodecyl sulfate (SDS) micellar solution. Time-resolved fluorescence measurements show that the fluorescence decays biexponentially in the micellar solution, indicating the relaxation of micellar environments surrounding the excited BXP. The major component of fluorescence lifetimes in CTAB or Triton X-100 micellar phase is even shorter (330–427ps) than in SDS micellar phase (731 ps). The nonradiative decay constants are significantly larger (ca 3.0 times 10⁹ s^?1) in the CTAB or Triton X-100 micellar phase than in SDS micelles by a factor of ca 10. The major nonradiative decay is interpreted to be the internal conversion due to nuclear geometric change of BXP in the first excited singlet state. This is consistent with the observation that the quantum yields of intersystem crossing are very low (less than 0.01) in the micellar solutions as determined by the laser flash photolysis technique. The laser-induced transient absorption spectrum of BXP in CTAB or Triton X-100 micellar solution shows that the decay kinetics of the transients in CTAB or Triton X-100 are significantly different from first order kinetics in SDS.     

Excited-state dynamics of donor-acceptor bridged systems containing a boron-dipyrromethene chromophore: interplay between charge separation and reorientational motion

The excited-state dynamics of a series of electron donor-acceptor bridged systems (DABS) consisting of a boron-dipyrromethene chromophore covalently linked to a dinitro-substituted triptycene has been investigated using femtosecond time-resolved spectroscopy. The chromophores differ by the number of bromine atom substituents. The fluorescence lifetime of the DABS without any bromine atom is strongly reduced when going from toluene to polar solvents, this shortening being already present in chloroform. This effect is about 10 times weaker with a single bromine atom and negligible with two bromine atoms on the chromophore. The excited-state lifetime shortening is ascribed to a charge transfer from the excited chromophore to a nitrobenzene moiety, the driving force of this process depending on the number of bromine substituents. The occurrence of this process is further confirmed by the investigation of the excited-state dynamics of the chromophore alone in pure nitrobenzene. Surprisingly, no correlation between the charge separation time constant and the dielectric properties of the solvents could be observed. However, a good correlation between the charge separation time constant and the diffusional reorientation time of the chromophore alone, measured by fluorescence anisotropy, was found. Quantum chemistry calculations suggest that quasi-free rotation about the single bond linking the chromophore to the triptycene moiety permits a sufficient coupling of the donor and the acceptor to ensure efficient charge separation. The charge separation dynamics in these molecules is thus controlled by the reorientational motion of the donor relative to the acceptor.     

(Sub)-picosecond spectral evolution of fluorescence in photoactive proteins studied with a synchroscan streak camera system

The spectral evolution of three photoactive proteins has been investigated by measuring the fluorescence with good temporal and wavelength resolution and a high signal-to-noise ratio. Upon excitation at 400 nm wild-type (wt) PYP both at neutral pH and in the low-pH blueshifted pBdark state exhibited a strong quenching of the fluorescence, the major part of which could be described by lifetimes of about 1.7 and 7.7 ps. The remaining fluorescence decay occurred multiexponentially with lifetimes between 30 and 125 ps. Additionally, in wtPYP at neutral pH, a dynamic Stokes shift was found to occur with a time constant of about 0.25 ps. In a PYP preparation that was reconstituted with the chromophore 7-hydroxy-coumarin-3- carboxylic acid rather than the native coumaric acid, and which is therefore not capable of performing the cis-trans-isomerization that initiates the photocycle in wtPYP, the fluorescence was found to decay multiexponentially with lifetimes of 51 ps, 0.33 and 3.77 ns. Additionally, dynamic Stokes shifts were observed with time constants of about 0.1 and 3.5 ps. Upon comparison of the dynamics of this preparation with that of wtPYP the multiexponential decay with lifetimes of 1.7 and 7.7 ps found in wtPYP was attributed to photochemistry of the p-coumaric-acid chromophore. The emission from bacteriorhodopsin mutant D85S upon excitation at 635 nm decays biexponentially with estimated lifetimes of 5.2 and 19.1 ps. No dynamic Stokes shift was observed here. Four lifetimes were needed to describe the decay of the emission from the A* state in the green fluorescent protein. From a target analysis it was concluded that the longer lifetimes are accompanied by a decreasing probability of forming I*, which approaches zero with the longest A* lifetime of 1.5 ns. These observations may be explained by heterogeneity of A and by relaxation of A*. In all three systems studied, multiexponential decay of emission was present, suggesting that heterogeneity is a common feature of these chromophore protein complexes.