Time-resolved Anisotropy Measurements Research Articles

Fluorescence investigations of the conformational behaviour of Poly( N-vinylcaprolactam)

Fluorescence techniques, particularly time-resolved anisotropy measurements, TRAMS, have confirmed that poly( N-vinylcaprolactam), PNVCap, undergoes a conformational transition from an expanded coil to a compact globule at its lower critical solution temperature, LCST. TRAMS and cloud point measurements have revealed the onset of the LCST as 38 °C. The nature of the conformational change for PNVCap is distinct from that of another thermoresponsive polymer, poly( N-isopropylacrylamide), PNIPAM: the discontinuity in the thermal dependence of the rate of macromolecular motion occurs over a range of ca. 15 °C for PNVCap while that for PNIPAM takes place between 31 and 33 °C. Furthermore, there is a reduction in the magnitude of the conformational change of PNVCap when compared to that of PNIPAM. Pyrene solubilisation experiments provide support for a model for PNVCap above the LCST, consisting of a globular structure with a hydrophobic core which is stabilised by more hydrophilic units on the surface.

Spectroscopic study of the chromophore–protein association and primary photoinduced events in the photoreceptor of Blepharisma japonicum

Blepharisma japonicum is a ciliated protozoan exhibiting a strong step-up photophobic response upon illumination. The photoreceptor chromophores responsible for this response have been identified to be hypericin-like chromophores (blepharismin and oxyblepharismin), complexed to a 200 kDa non-water-soluble protein. The present work opens up new perspectives on the primary phototransduction steps of B. japonicum's light perception through a joined approach by steady-state fluorescence spectroscopy, time-resolved fluorescence anisotropy and sub-picosecond transient absorption spectroscopy. The free chromophore of the light-adapted form of the cell (oxyblepharismin) was studied in various solvents and its spectroscopic properties, as well as its primary excited-state reactivity, compared with those of the corresponding pigment-protein complex, extracted by phosphate-concentration-step chromatography on a hydroxyapatite column. Fluorescence anisotropy together with SDS PAGE electrophoresis results confirm that oxyblepharismin is non-covalently bound to the apoprotein and show that, in the excited state, it is free to rotate in all directions within the binding site where it experiences a large local viscosity. Time-resolved anisotropy measurements on aromatic amino acids confirm that the molecular weight of the protein is of the order of 200 kDa. Although showing very similar steady-state spectra, free oxyblepharismin and its protein complex have noticeably different excited-state behaviours. In particular, the protein complex exhibits a pronounced short-lived absorption feature in the 640--750 nm range, decaying biexponentially in 4 ps and 56 ps. Those decays, also observed in other spectral regions, are not found in the corresponding kinetics of the isolated pigment in solution. This early behaviour of the protein complex might be the signature of the primary phototransduction process, possibly involving an electron transfer from the pigment to a neighbouring protein acceptor residue as it had been suggested in previous studies.

Luminescence anisotropy-based detection of nucleic acids and proteins using long-lifetime Ru(II) complex as a luminescent label

Luminescence anisotropy-based methods are powerful tool for the detection of biomolecules in homogeneous physiological media without Bound/Free separation. However, analyses based on the method are always disturbed by the autofluorescence from biological specimen. This disturbance limits the sensitivity of the method. To improve the detection limit of the method, a long-lifetime luminophore was adopted as the label. Because of the lifetime of the autofluorescence is below 10 ns, it is expected that the use of the long-lifetime luminophore enables us to avoid the disturbance by adopting time-resolved luminescence anisotropy measurement. To verify this concept, oligodeoxyribonucleotides and Staphylococcus aureus protein A were labeled with Ru(II) complex (tau approximately 500 nsec) and used for the detection of Escherichia coli 16S ribosomal RNA (16S rRNA) and immunoglobulin G (IgG), respectively. Results indicated that the methods were useful for the detection of rRNA and IgG without disturbance of the autofluorescence.

Single-spanning membrane protein insertion in membrane mimetic systems: role and localization of aromatic residues

Membrane protein insertion in the lipid bilayer is determining for their activity and is governed by various factors such as specific sequence motifs or key amino-acids. A detailed fluorescence study of such factors is exemplified with PMP1, a small (38 residues) single-membrane span protein that regulates the plasma membrane H(+)-ATPase in yeast and specifically interacts with phosphatidylserines. Such interactions may stabilize raft domains that have been shown to contain H(+)-ATPase. Previous NMR studies of various fragments have focused on the critical role of interfacial residues in the PMP1 structure and intermolecular interactions. The C-terminal domain contains a terminal Phe (F38), a single Trp (W28) and a single Tyr (Y25) that may act together to anchor the protein in the membrane. In order to describe the location and dynamics of W28 and the influence of Y25 on protein insertion within membrane, we carried out a detailed steady-state and time-resolved fluorescence study of the synthetic G13-F38 fragment and its Tyr-less mutant, Y25L in various membrane mimetic systems. Detergent micelles are conveniently used for this purpose. We used dodecylphosphocholine (DPC) in order to compare with and complement previous NMR results. In addition, dodecylmaltoside (DM) was used so that we could apply our recently described new quenching method by two brominated analogs of DM (de Foresta et al. 2002, Eur. Biophys. J. 31:185-97). In both systems, and in the presence and absence of Y25, W28 was shown to be located below but close to the polar headgroup region, as shown by its maximum emission wavelengths (lambda(max)), curves for the quenching of Trp by the brominated analogs of DM and bimolecular constants for quenching (k(q)) by acrylamide. Results were interpreted by comparison with calibration data obtained with fluorescent model peptides. Time-resolved anisotropy measurements were consistent with PMP1 fragment immobilization within peptide-detergent complexes. We tentatively assigned the two major Trp lifetimes to the Trp (chi(1)=60 degrees and 180 degrees ) rotamers, based on the recent lifetime-rotamer correlation proposed for model cyclic peptides (Pan and Barkley 2004, Biophys J 86:3828-35). We also analyzed the role of the hydrophobic anchor, by comparing the micelle binding of fragments of various lengths including the synthesized full-length protein and detected peculiar differences for protein interaction with the polar headgroups of DM or DPC.

2‐5A induces a conformational change in the ankyrin‐repeat domain of RNase L

RNase L is responsible for the 2-5A host defense system, an RNA degradation pathway present in cells of higher vertebrates that functions in both the antiviral and anticellular activities of interferon. The activity of RNase L is tightly regulated and is exerted only in the presence of 2-5A. The postulated mechanism of its regulation is as follows: the N-terminal half ankyrin-repeat domain masks the C-terminal half nuclease domain in the absence of 2-5A. On binding 2-5A at the ankyrin-repeat domain, RNase L forms a homodimer and removes the ankyrin-repeat domain from the nuclease domain to become the active form. A conformational change in the ankyrin-repeat domain is a key step in this hypothetical mechanism, but there is as yet no evidence for such a change. To clarify the events induced by 2-5A binding, we established procedures for expression and purification of the ankyrin-repeat domain of human RNase L. Fluorescence spectra of the protein showed clear difference in the presence and absence of 2-5A. The alterations in the spectra supported conformational changes of the protein. Time-resolved anisotropy measurements indicated that 2-5A binding led to a significant decrease in the rotational radius of the protein. In addition, 2-5A provided the domain with resistance to protease digestion as a result of a conformational change. These results indicated that the ankyrin-repeat domain of RNase L constricts its structure by binding of 2-5A. This observation suggests a revised model of the 2-5A-induced activation of RNase L.

Ultrafast relaxation dynamics of a biologically relevant probe dansyl at the micellar surface

We report picosecond-resolved measurement of the fluorescence of a well-known biologically relevant probe, dansyl chromophore at the surface of a cationic micelle (cetyltrimethylammonium bromide, CTAB). The dansyl chromophore has environmentally sensitive fluorescence quantum yields and emission maxima, along with large Stokes shift. In order to study the solvation dynamics of the micellar environment, we measured the fluorescence of dansyl chromophore attached to the micellar surface. The fluorescence transients were observed to decay (with time constant ∼350 ps) in the blue end and rise with similar timescale in the red end, indicative of solvation dynamics of the environment. The solvation correlation function is measured to decay with time constant 338 ps, which is much slower than that of ordinary bulk water. Time-resolved anisotropy of the dansyl chromophore shows a bi-exponential decay with time constants 413 ps (23%) and 1.3 ns (77%), which is considerably slower than that in free solvents revealing the rigidity of the dansyl-micelle complex. Time-resolved area-normalized emission spectroscopic (TRANES) analysis of the time dependent emission spectra of the dansyl chromophore in the micellar environment shows an isoemissive point at 21066 cm −1. This indicates the fluorescence of the chromophore contains emission from two kinds of excited states namely locally excited state (prior to charge transfer) and charge transfer state. The nature of the solvation dynamics in the micellar environments is therefore explored from the time-resolved anisotropy measurement coupled with the TRANES analysis of the fluorescence transients. The time scale of the solvation is important for the mechanism of molecular recognition.

Fluorescence depolarization in the presence of excitation energy migration in partly ordered polymer films

Excitation energy migration is studied for 3,3′-diethylthiacyanine iodide in uniaxially stretched and unstretched poly(vinyl alcohol) films. Time-resolved and steady-state measurements of fluorescence depolarization yield quite different results for disordered and partly ordered systems. Contrary to disordered systems, for which strong concentration depolarization was observed, partly ordered systems exhibit much weaker depolarization as a result of preferential angular distribution of transition dipole moments of identical fluorophores participating in energy migration. Difference of the same origin was found for time-resolved emission anisotropy measurements. These results are in agreement with those obtained from the Monte-Carlo simulation.

Quantitative Analysis of the Fluorescence Properties of Intrinsically Fluorescent Proteins in Living Cells

The main potential of intrinsically fluorescent proteins (IFPs), as noninvasive and site-specific markers, lies in biological applications such as intracellular visualization and molecular genetics. However, photophysical studies of IFPs have been carried out mainly in aqueous solution. Here, we provide a comprehensive analysis of the intracellular environmental effects on the steady-state spectroscopy and excited-state dynamics of green (EGFP) and red (DsRed) fluorescent proteins, using both one- and two-photon excitation. EGFP and DsRed are expressed either in the cytoplasm of rat basophilic leukemia (RBL-2H3) mucosal mast cells or anchored (via LynB protein) to the inner leaflet of the plasma membrane. The fluorescence lifetimes (within ∼10%) and spectra in live cells are basically the same as in aqueous solution, which indicate the absence of both IFP aggregation and cellular environmental effects on the protein folding under our experimental conditions. However, comparative time-resolved anisotropy measurements of EGFP reveal a cytoplasmic viscosity 2.5±0.3 times larger than that of aqueous solution at room temperature, and also provide some insights into the LynB-EGFP structure and the heterogeneity of the cytoplasmic viscosity. Further, the oligomer configuration and internal depolarization of DsRed, previously observed in solution, persists upon expression in these cells. DsRed also undergoes an instantaneous three-photon induced color change under 740-nm excitation, with efficiently nonradiative green species. These results confirm the implicit assumption that in vitro fluorescence properties of IFPs are essentially valid for in vivo applications, presumably due to the β-barrel protection of the embodied chromophore. We also discuss the relevance of LynB-EGFP anisotropy for specialized domains studies in plasma membranes.

Ultrafast Relaxation Dynamics from the S2 State of Malachite Green Studied with Femtosecond Upconversion Spectroscopy

We have studied the relaxation dynamics in a triphenyl methane (TPM) dye, malachite green (MG), following S2-state excitation using fluorescence up-conversion measurements in ethanol and ethylene glycol solutions. Information on the mechanisms and dynamics of radiationless transitions from higher excited states in TPM dyes is interesting from the torsional dynamic aspects of the phenyl rings and for the applications of TPM dyes involving knowledge of the excited-state relaxation channels. Kinetic measurements at different wavelengths suggested a cascade population relaxation along the S2 state with a time constant of ∼130 fs that is almost independent of solvent viscosity in the two solvents used, contrary to the relaxation dynamics of the S1 state. Wavelength-dependent time-resolved anisotropy measurements along the S2 and S1 emission bands displayed successive reductions in the anisotropy values, indicating a continuous evolution to the S1 surface. A detailed analysis led us to propose a relaxation path...

Steady-state and time-resolved fluorescence studies of conformational changes induced by cyclic AMP and DNA binding to cyclic AMP receptor protein from Escherichia coli.

cAMP receptor protein (CRP), allosterically activated by cAMP, regulates the expression of several genes in Escherichia coli. As binding of cAMP leads to undefined conformational changes in CRP, we performed a steady-state and time-resolved fluorescence study to show how the binding of the ligand influences the structure and dynamics of the protein. We used CRP mutants containing a single tryptophan residue at position 85 or 13, and fluorescently labeled with 1,5-I-AEDANS attached to Cys178. Binding of cAMP in the CRP-(cAMP)2 complex leads to changes in the Trp13 microenvironment, whereas its binding in the CRP-(cAMP)4 complex alters the surroundings of Trp85. Time-resolved anisotropy measurements indicated that cAMP binding in the CRP-(cAMP)2 complex led to a substantial increase in the rotational mobility of the Trp13 residue. Measurement of fluorescence energy transfer (FRET) between labeled Cys178 and Trp85 showed that the binding of cAMP in the CRP-(cAMP)2 complex caused a substantial increase in FRET efficiency. This indicates a decrease in the distance between the two domains of the protein from 26.6 A in apo-CRP to 18.7 A in the CRP-(cAMP)2 complex. The binding of cAMP in the CRP-(cAMP)4 complex resulted in only a very small increase in FRET efficiency. The average distance between the two domains in CRP-DNA complexes, possessing lac, gal or ICAP sequences, shows an increase, as evidenced by the increase in the average distance between Cys178 and Trp85 to approximately 20 A. The spectral changes observed provide new structural information about the cAMP-induced allosteric activation of the protein.

Time-Resolved Fluorescence Anisotropy Measurements Made Simple

Time-resolved anisotropy measurements were performed using simple instrumentation with the aim to demonstrate the speed and ease of the experiment. Subsequent data analysis examples involved common, easily adaptable procedures.

Role of protein and substrate dynamics in catalysis by Pseudomonas putida cytochrome P450cam.

The role of protein structural flexibility and substrate dynamics in catalysis by cytochrome P450 enzymes is an area of current interest. We have addressed these in cytochrome P450(cam) (P450(cam)) and its Y96A mutant with camphor and its related compounds using fluorescence spectroscopy. Previously [Prasad et al. (2000) FEBS Lett. 477, 157-160], we provided experimental support to dynamic fluctuations in P450(cam), and substrate access into the active site region via the channel next to the flexible F-G helix-loop-helix segment. In the investigation described here, we show that the dynamic fluctuations in the enzyme are substrate dependent as reflected by tryptophan fluorescence quenching experiments. The orientation of tryptophan relative to heme (kappa(2)) for W42 obtained from time-resolved tryptophan fluorescence measurements show variation with type of substrate bound to P450(cam) suggesting regions distant from heme-binding site are affected by physicochemical and steric characteristics/protein-substrate interactions of P450(cam) active site. We monitored substrate dynamics in the active site region of P450(cam) by time-resolved substrate anisotropy measurements. The anisotropy decay of substrates bound to P450(cam) indicate that mobility of substrates is modulated by physicochemical and steric characteristics/protein-substrate interactions of local active site structure, and provides an understanding of factors controlling observed hydroxylated products for substrate bound P450(cam) complexes. The present study shows that P450(cam) local and peripheral structural flexibility and heterogeneity along with substrate mobility play an important role in regulating substrate binding orientation during catalysis and accommodating diverse range of substrates within P450(cam) heme pocket.

Phosphorylation induces a conformational transition near the lipid-water interface of phospholamban reconstituted with the Ca-ATPase.

We have measured conformational changes of phospholamban (PLB) induced both by its interaction with the SR Ca-ATPase and by phosphorylation of Ser-16 by cAMP-dependent protein kinase (PKA) using an engineered PLB having a single cysteine (Cys-24) derivatized with the fluorophore 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (ANSmal). This modified mutant PLB is fully functional when co-reconstituted with the affinity-purified Ca-ATPase in liposomes. ANSmal emission properties and its solvent accessibility indicate that Cys-24 is in an aqueous environment outside the membrane. Fluorescence quenching and time-resolved anisotropy measurements of ANSmal-PLB demonstrate distinct structures for PLB in the free and Ca-ATPase-bound state. Both solvent exposure and probe motions of ANSmal are enhanced upon interaction of PLB with the Ca-ATPase. This conformational transition entails conversion of free PLB in a conformation which is insensitive to one which is sensitive to the phosphorylation state of PLB. Upon phosphorylation of Ca-ATPase-bound PLB, a decreased level of solvent exposure of ANSmal is observed, suggesting that the amino acid sequence of PLB near the lipid-water interface acts as a conformational switch in response to the phosphorylation of PLB. A longer correlation time, resolved by anisotropy measurements, corresponding to polypeptide chain fluctuations, is substantially restricted by interaction of PLB with the Ca-ATPase. This restriction is not reversed by phosphorylation of PLB, indicating that the region around Cys-24 near the lipid-water interface does not undergo dissociation from the Ca-ATPase. These results suggest that the phosphorylation by PKA induces a redistribution of PLB-Ca-ATPase protein contacts to relieve the inhibitory effect of PLB for the activation of calcium transport.

Ultrafast structural transformations in graphite

Femtosecond laser-induced structural transitions in graphite were studied by time-resolved optical anisotropy measurements. The decay of the anisotropic reflectivity seems to indicate a loss of long-range order on a subpicosecond time scale, which is much faster than the electron-phonon relaxation time. This observation confirms the nonthermal nature of the structural transition.

A series of polyrotaxanes containing α-cyclodextrin and naphthalene-modified α-cyclodextrin and solvent effects on the fluorescence quenching by terminal units

Photoquenching systems were constructed using polyrotaxanes in which cyclodextrin (CD) rings of α-CD and 6-deoxy-6-(-naphthalene-2-sulfonyl)-α-cyclodextrin (NpCD) are threaded by a poly(ethylene glycol) chain with trinitrophenyl (acceptor) units at both ends (8–12). The effects of the component ratio of the polyrotaxanes on the naphthalene fluorescence quenching efficiency and energy migration between naphthalene units were examined by steady-state and time-resolved spectra and anisotropy measurements. The quenching efficiency of 8, which has the lowest content of NpCD, is larger than those of the polyrotaxanes with higher NpCD contents; however this tendency depends on the solvents. We explain the quenching mechanism of naphthalene fluorescence by the combination process of Forster type energy transfer and electron transfer to the trinitrophenyl units.

Ultrafast Rotational Anisotropy Measurements: Strong-Field Nonlinear Saturation Effects

Nonlinear saturation effects in ultrafast time-resolved rotational anisotropy measurements are explored as a function of increased pump laser intensity. Femtosecond pump−probe experimental data were obtained over 3 orders of magnitude in pump laser intensity ranging from no saturation to highly nonlinear saturation. Data were obtained for molecular iodine vapor at room temperature following resonant excitation of the B←X transition. For low pump laser intensities, the data are shown to fit the conventional anisotropy formalism for unidirectional detection well. For higher pump laser intensities, the overall anisotropy diminishes because of saturation and the conventional fits diverge from the experimental data. In this regime, a saturation parameter can be introduced in the formulation to improve the fit between the model and the experimental data, thereby improving the accuracy of the resulting rotational temperatures. At the highest laser intensities, an additional photochemical pathway arising from the...

Fluorescence Anisotropy: A Method for Early Detection of Alzheimer β-Peptide (Aβ) Aggregation

Time-resolved anisotropy measurements (TRAMS) have been used to study the aggregation of the β-amyloid (Aβ) peptide which is suspected of playing a central role in the pathogenesis of Alzheimer's Disease (AD). The experiments, which employ small quantities of fluorescently-labelled Aβ, in addition to the untagged peptide, have shown that the sensitive TRAMS technique detects the presence of preformed “seed” particles in freshly prepared solutions of Aβ. More importantly, as 100 μM solutions of Aβ containing tagged Aβ at a concentration level of either 0.5 or 1 μM are incubated, the TRAMS prove capable of detection of the peptide aggregation process through the appearance of a continuously increasing “residual anisotropy” within the time-resolved fluorescence data. The method detects Aβ aggregation in its earliest stages, well before complexation becomes apparent in more conventional methods such as the thioflavin T fluorescence assay. The TRAMS approach promises to provide a most attractive route for establishment of a high-throughput procedure for the early detection of the presence of amyloid aggregates in the screening of biological samples.

Fluorescence investigations of the thermally induced conformational transition of poly( N-isopropylacrylamide)

Poly( N-isopropylacrylamide), PNIPAM, undergoes phase separation, upon heating under semi-dilute conditions, at 32°C, the lower critical solution temperature (LCST). Upon subsequent cooling, the dispersion rapidly clears again, at the LCST, to form a single, homogeneous phase. In this paper we report that fluorescence techniques, including quenching and time-resolved anisotropy measurements (TRAMS) on ultra-dilute (10 −3 wt%) aqueous solutions of an acenaphthylene labelled sample, provide conclusive evidence that a coil collapse mechanism is implicated in the thermoreversible behaviour: the polymer undergoes a conformational switch from an open coil below 32°C to a compact, globular structure above the LCST. TRAMS reveal that a marked reduction in the segmental mobility of the polymer occurs at the onset of the LCST. In addition, a dramatic change in the accessibility of the label to aqueous-borne quenchers is also apparent at temperatures in excess of 32°C. PNIPAM is capable of solubilising low molar mass species in its compact form: changes in the vibrational fine structure of the emission spectrum of pyrene have been used to monitor uptake and release of the probe. Excited state lifetime measurements have also proven to be sensitive monitors of the conformational switch of PNIPAM: at temperatures greater than 32°C, τ increases to ca. 160 ns which is indicative of pyrene sequestered in a hydrophobic, protective environment. Release of the probe into the aqueous phase results in a dramatic reduction of τ to ca. 130 ns which is characteristic of pyrene dispersed in water. These data highlight the potential of NIPAM based polymers to act as carriers in controlled release applications. Fluorescence techniques have proved capable of monitoring changes in chain mobility, the degree of coil compaction and solubilization capacity (for organic guests) of PNIPAM as it is raised through its LCST. Information of this type is not readily obtained through other techniques and fluorescence approaches should prove invaluable in future investigations of the effects of chemical modification in attempts to manipulate the LCST of NIPAM-based systems.

The slow folding reaction of barstar: the core tryptophan region attains tight packing before substantial secondary and tertiary structure formation and final compaction of the polypeptide chain

The slow folding of a single tryptophan-containing mutant of barstar has been studied in the presence of 2 M urea at 10°C, using steady state and time-resolved fluorescence methods and far and near-UV CD measurements. The protein folds in two major phases: a fast phase, which is lost in the dead time of measurement during which the polypeptide collapses to a compact form, is followed by a slow observable phase. During the fast phase, the rotational correlation time of Trp53 increases from 2.2 ns to 7.2 ns, and its mean fluorescence lifetime increases from 2.3 ns to 3.4 ns. The fractional changes in steady-state fluorescence, far-UV CD, and near-UV CD signals, which are associated with the fast phase are, respectively, 36 %, 46 %, and 16 %. The product of the fast phase can bind the hydrophobic dye ANS. These observations together suggest that the folding intermediate accumulated at the end of the fast phase has: (a) about 20 % of the native-state secondary structure, (b) marginally formed or disordered tertiary structure, (c) a water-intruded and mobile protein interior; and (d) solvent-accessible patches of hydrophobic groups. Measurements of the anisotropy decay of Trp53 suggest that it undergoes two types of rotational motion in the intermediate: (i) fast (τr≈1 ns) local motion of its indole side-chain, and (ii) a slower (τr≈7.2 ns) motion corresponding to global tumbling of the entire protein molecule. The ability of the Trp53 side-chain to undergo fast local motion in the intermediate, but not in the fully folded protein where it is completely buried in the hydrophobic core, suggests that the core of the intermediate is still poorly packed. The global tumbling time of the fully folded protein is faster at 5.6 ns, suggesting that the volume of the intermediate is 25 % more than that of the fully folded protein. The rate of folding of this intermediate to the native state, measured by steady-state fluorescence, far-UV CD, and near-UV CD, is 0.07(±0.01) min−1 This rate compares to a rate of folding of 0.03(±0.005) min−1, determined by double-jump experiments which monitor directly formation of native protein; and to a rate of folding of 0.05 min−1, when determined from time-resolved anisotropy measurements of the long rotational correlation time, which relaxes from an initial value of 7.2 ns to a final value of 5.6 ns as the protein folds. On the other hand, the amplitude of the short correlation time decreases rapidly with a rate of 0.24(±0.06) min−1. These results suggest that tight packing of residues in the hydrophobic core occurs relatively early during the observable slow folding reaction, before substantial secondary and tertiary structure formation and before final compaction of the protein.

Ultrafast electron localization dynamics following photo-induced charge transfer

Molecular dynamics occurring in the earliest stages following photo-induced charge transfer were investigated. Femtosecond time-resolved absorption anisotropy measurements on [Ru(bpy)(3)](2+), where bpy is 2,2'-bipyridine, reveal a time dependence in nitrile solutions attributed to initial delocalization of the excited state over all three ligands followed by charge localization onto a single ligand. The localization process is proposed to be coupled to nondiffusive solvation dynamics. In contrast, measurements sampling population dynamics show spectral evolution associated with wave packet motion on the excited state surface that is independent of solvent. The results therefore reveal two important contributions to the evolution of charge transfer states in condensed phase, one that is strongly coupled to the surrounding environment and another that follows a potential internal to the molecule.