Ultrafast Fluorescence Spectroscopy Research Articles

The energy transfer dynamics in C-phycocyanin hexamer from <italic>Spirulina platensis</italic>

Photosynthesis is an ancient and important conversion of solar light to biological energy in photosynthetic organisms. Phycobilisome (PBS) system in cyanobacteria and red algae is one of two major light-harvesting systems of photosynthetic oxygen organisms. PBS is aggregation of water-soluble phycobiliproteins (PBPs) which are distinctively colored group of disk-shaped macromolecular proteins bearing covalently attached open-chain tetrapyrroles known as phycobilins. PBPs are capable to absorb solar light and transfer energy to the chlorophyll of photosynthetic reaction center with a high efficiency. Light energy absorbed by phycobilins is transmitted between the subunits, and then transmitted between different PBPs, finally to the photoreaction center located on the thylakoid membrane. The energy transfer between PBPs is very fast and high efficiency. Because energy can be transferred in different chromophores and there are a large number of chromophores in PBPs, the energy transfer path of PBPs is complex and diverse. In the past 20 years, energy transfer process of PBPs have been analyzed by time-resolved fluorescence spectroscopy because a large shift of the fluorescence peaks can be clearly observed. For example, Zhang et al. estimated the time constant for energy transfer from PC (rod) to APC (core) as 18 ps and 55 ps based on PBPs crystal structures and time-resolved fluorescence spectra of the rod-core complex. However, due to the technical bottleneck of theoretical simulation and spectral experimental detection, the research on the physical mechanism of photosynthetic energy transfer is relatively slow. In recent years, the ultrafast spectrum time resolution has been continuously improved, which provides the conditions for studying the energy transfer of PBPs. In this paper, the mechanism of energy transfer in C-phycocyanin hexamer from Spirulina platensis was studied by ultrafast time-resolved fluorescence spectroscopy. The energy transfer pathway and the related transfer parameters in the C-phycocyanin hexamer were identified by the fitting of the fluorescence decay curve at different detection wavelengths. It was proved that the energy transfer of C-phycocyanin hexamer had four time components from the experiment, which are 6, 22, 280 and 1470 ps, respectively, then the time components were identified and discussed. This study reflects the energy transfer kinetics between different chromophores in C-phycocyanin and provides important experimental data for solving the relationship between the structure and function of PBPs. At the same time, our study has important value for the understanding of the primary process of photosynthesis. In the future, if the resolution of a streak camera can be raised to femtoseconds, it is possible to find more energy transfer paths, and thus it is expected to make a breakthrough in the ultrafast and ultra-efficient energy transfer mechanism of PBPs.

Prying into Hydration Water in Amyloidogenic Intrinsically Disordered Proteins

The hydration water layer, termed as ‘biological water’, present at the protein-water interface shares an intimate relationship with protein functions. However, little is known about hydration water in intrinsically disordered proteins (IDPs) that are capable of adopting a multitude of conformational states. Recent studies have revealed that many IDPs, due to their astonishing conformational plasticity, can readily convert into highly ordered amyloid aggregates that are associated with several deadly human diseases. For instance, α-synuclein is an IDP present in nerve cells and aggregates into amyloids via toxic oligomeric intermediates in Parkinson's disease. To address the role of water in the pathological conversion of α-synuclein, we utilized a combination of ultrafast measurements and all-atom molecular dynamics simulations. We created a number of single cysteine mutants and labeled these variants using environment-sensitive thiol-active fluorophore. We then monitored the solvation dynamics using ultrafast fluorescence spectroscopy that is capable of capturing the femtosecond time-resolved snapshots of water molecules around the polypeptide chain. These site-specific ultrafast studies allowed us to construct the hydration map of α-synuclein. Our studies revealed the presence of three distinct types of water: bulk-, hydration- and confined water. Atomistic molecular dynamics simulations supported our experimental observations and provided insights into the molecular origin of confined water. Our results from this experimental-simulation approach reveal trapping of interfacial water near the amyloidogenic hotspot that triggers the conversion of IDPs into pathological amyloids via entropically favorable water exclusion events during disorder-to-order amyloid transition. Our findings provide an experimental underpinning of significant water rearrangements associated with both chain desolvation and water confinement upon amyloid formation from IDPs. I will also discuss our new results on the hydration dynamics of a diverse range of amyloidogenic IDPs possessing different conformational preferences due to varied chain-chain and chain-solvent interactions.

Identification of the TyrOH•+ Radical Cation in the Flavoenzyme TrmFO.

Tyrosine (TyrOH) and tryptophan radicals play important roles as intermediates in biochemical charge-transfer reactions. Tryptophanyl radicals have been observed both in their protonated cation form and in their unprotonated neutral form, but to date, tyrosyl radicals have only been observed in their unprotonated form. With a genetically modified form of the flavoenzyme TrmFO as a suitable model system and using ultrafast fluorescence and absorption spectroscopy, we characterize its protonated precursor TyrOH•+, and we show this species to have a distinct visible absorption band and a transition moment that we suggest to lie close to the phenol symmetry axis. TyrOH•+ is formed in ∼1 ps by electron transfer to excited flavin and decays in ∼3 ps by charge recombination. These findings imply that TyrOH oxidation does not necessarily induce its concerted deprotonation. Our results will allow disentangling of photoproduct states in flavoproteins in often-encountered complex situations and more generally are important for understanding redox chains relying on tyrosyl intermediates.

Drastic difference between hole and electron injection through the gradient shell of CdxSeyZn1-xS1-y quantum dots.

Ultrafast fluorescence spectroscopy was used to investigate the hole injection in CdxSeyZn1-xS1-y gradient core-shell quantum dot (CSQD) sensitized p-type NiO photocathodes. A series of CSQDs with a wide range of shell thicknesses was studied. Complementary photoelectrochemical cell measurements were carried out to confirm that the hole injection from the active core through the gradient shell to NiO takes place. The hole injection from the valence band of the QDs to NiO depends much less on the shell thickness when compared to the corresponding electron injection to n-type semiconductor (ZnO). We simulate the charge carrier tunneling through the potential barrier due to the gradient shell by numerically solving the Schrödinger equation. The details of the band alignment determining the potential barrier are obtained from X-ray spectroscopy measurements. The observed drastic differences between the hole and electron injection are consistent with a model where the hole effective mass decreases, while the gradient shell thickness increases.

Retardation of Bulk Water Dynamics by Disaccharide Osmolytes.

The bioprotective nature of disaccharides is hypothesized to derive from the modification of the hydrogen bonding network of water which protects biomolecules through lowered water activity at the protein interface. Using ultrafast fluorescence spectroscopy, we measured the relaxation of bulk water dynamics around the induced dipole moment of two fluorescent probes (Lucifer Yellow Ethylenediamine and Tryptophan). Our results indicate a reduction in bulk water reorganization rate of approximately 30%. We observe this retardation in the low concentration regime measured at 0.1 and 0.25 M, far below the onset of glassy dynamics. This reduction in water activity could be significant in crowded biological systems, contributing to global change in protein energy landscape, resulting in a significant enhancement of protein stability under environmental stress. We observed similar dynamic reduction for two disaccharide osmolytes, sucrose and trehalose, with trehalose being the more effective in reducing solvation dynamics.

Evolution of Nonmirror Image Fluorescence Spectra in Conjugated Polymers and Oligomers.

The nonmirror image relationship between absorption and fluorescence spectra of conjugated polymers contrasts with most organic chromophores and is widely considered a signature of interchromopohore energy funneling. We apply broad-band ultrafast fluorescence spectroscopy to resolve the evolution of fluorescence spectra for dilute solutions of conjugated oligothiophenes, where no energy transfer is possible. Fluorescence spectra evolve from a mirror image of absorption, which lacks vibronic structure, toward a spectrally narrower and vibronically structured species on the hundreds of femtosecond to early picosecond time scale. Our analysis of this fluorescence spectral evolution shows that a broad distribution of torsional conformers is driven to rapidly planarize in the excited state, including in solid films, which is supported by Raman spectroscopy and quantum chemical modeling. Our data have important implications for understanding different energy-transfer regimes that are delineated by structural relaxation.

Excitation relaxation dynamics and energy transfer in pigment-protein complexes of a dinoflagellate, revealed by ultrafast fluorescence spectroscopy.

Photosynthetic light-harvesting complexes, found in aquatic photosynthetic organisms, contain a variety of carotenoids and chlorophylls. Most of the photosynthetic dinoflagellates possess two types of light-harvesting antenna complexes: peridinin (Peri)-chlorophyll (Chl) a/c-protein, as an intrinsic thylakoid membrane complex protein (iPCP), and water-soluble Peri-Chl a-protein, as an extrinsic membrane protein (sPCP) on the inner surface of the thylakoid. Peri is a unique carotenoid that has eight C=C bonds and one C=O bond, which results in a characteristic absorption band in the green wavelength region. In the present study, excitation relaxation dynamics of Peri in solution and excitation energy transfer processes of sPCP and the thylakoid membranes, prepared from the photosynthetic dinoflagellate, Symbiodinium sp., are investigated by ultrafast time-resolved fluorescence spectroscopy. We found that Peri-to-Chl a energy transfer occurs via the Peri S1 state with a time constant of 1.5ps or 400fs in sPCP or iPCP, respectively, and that Chl c-to-Chl a energy transfer occurs in the time regions of 350-400fs and 1.8-2.6ps.

Quantitative sampling of conformational heterogeneity of a DNA hairpin using molecular dynamics simulations and ultrafast fluorescence spectroscopy.

Molecular dynamics (MD) simulations and time resolved fluorescence (TRF) spectroscopy were combined to quantitatively describe the conformational landscape of the DNA primary binding sequence (PBS) of the HIV-1 genome, a short hairpin targeted by retroviral nucleocapsid proteins implicated in the viral reverse transcription. Three 2-aminopurine (2AP) labeled PBS constructs were studied. For each variant, the complete distribution of fluorescence lifetimes covering 5 orders of magnitude in timescale was measured and the populations of conformers experimentally observed to undergo static quenching were quantified. A binary quantification permitted the comparison of populations from experimental lifetime amplitudes to populations of aromatically stacked 2AP conformers obtained from simulation. Both populations agreed well, supporting the general assumption that quenching of 2AP fluorescence results from pi-stacking interactions with neighboring nucleobases and demonstrating the success of the proposed methodology for the combined analysis of TRF and MD data. Cluster analysis of the latter further identified predominant conformations that were consistent with the fluorescence decay times and amplitudes, providing a structure-based rationalization for the wide range of fluorescence lifetimes. Finally, the simulations provided evidence of local structural perturbations induced by 2AP. The approach presented is a general tool to investigate fine structural heterogeneity in nucleic acid and nucleoprotein assemblies.

Excited-state intramolecular proton transfer of 2-acetylindan-1,3-dione studied by ultrafast absorption and fluorescence spectroscopy.

We employ transient absorption from the deep-UV to the visible region and fluorescence upconversion to investigate the photoinduced excited-state intramolecular proton-transfer dynamics in a biologically relevant drug molecule, 2-acetylindan-1,3-dione. The molecule is a ß-diketone which in the electronic ground state exists as exocyclic enol with an intramolecular H-bond. Upon electronic excitation at 300 nm, the first excited state of the exocyclic enol is initially populated, followed by ultrafast proton transfer (≈160 fs) to form the vibrationally hot endocyclic enol. Subsequently, solvent-induced vibrational relaxation takes place (≈10 ps) followed by decay (≈390 ps) to the corresponding ground state.

Ultrafast excited-state dynamics and fluorescence deactivation of near-infrared fluorescent proteins engineered from bacteriophytochromes.

Near-infrared fluorescent proteins, iRFPs, are recently developed genetically encoded fluorescent probes for deep-tissue in vivo imaging. Their functions depend on the corresponding fluorescence efficiencies and electronic excited state properties. Here we report the electronic excited state deactivation dynamics of the most red-shifted iRFPs: iRFP702, iRFP713 and iRFP720. Complementary measurements by ultrafast broadband fluorescence and absorption spectroscopy show that single exponential decays of the excited state with 600 ~ 700 ps dominate in all three iRFPs, while photoinduced isomerization was completely inhibited. Significant kinetic isotope effects (KIE) were observed with a factor of ~1.8 in D2O, and are interpreted in terms of an excited-state proton transfer (ESPT) process that deactivates the excited state in competition with fluorescence and chromophore mobility. On this basis, new approaches for rational molecular engineering may be applied to iRFPs to improve their fluorescence.

Fluorescence kinetics of Trp–Trp dipeptide and its derivatives in water via ultrafast fluorescence spectroscopy

Ultrafast fluorescence dynamics of Tryptophan–Tryptophan (Trp–Trp/Trp2) dipeptide and its derivatives in water have been investigated using a picosecond resolved time correlated single photon counting (TCSPC) apparatus together with a femtosecond resolved upconversion spectrophotofluorometer. The fluorescence decay profiles at multiple wavelengths were fitted by a global analysis technique. Nanosecond fluorescence kinetics of Trp2, N-tert-butyl carbonyl oxygen-N′-aldehyde group-l-tryptophan-l-tryptophan (NBTrp2), l-tryptophan-l-tryptophan methyl ester (Trp2Me), and N-acetyl-l-tryptophan-l-tryptophan methyl ester (NATrp2Me) exhibit multi-exponential decays with the average lifetimes of 1.99, 3.04, 0.72 and 1.22ns, respectively. Due to the intramolecular interaction between two Trp residues, the “water relaxation” lifetime was observed around 4ps, and it is noticed that Trp2 and its derivatives also exhibit a new decay with a lifetime of ∼100ps, while single-Trp fluorescence decay in dipeptides/proteins shows 20–30ps. The intramolecular interaction lifetime constants of Trp2, NBTrp2, Trp2Me and NATrp2Me were then calculated to be 3.64, 0.93, 11.52 and 2.40ns, respectively. Candidate mechanisms (including heterogeneity, solvent relaxation, quasi static self-quenching or ET/PT quenching) have been discussed.

Excited-state vibrational relaxation and deactivation dynamics of trans-4-(N,N-dimethylamino)-4′-nitrostilbene in nonpolar solvents studied by ultrafast time-resolved broadband fluorescence spectroscopy

Abstract The excited-state relaxation dynamics of trans -4-( N , N -dimethylamino)-4′-nitrostilbene excited to the first absorption band in nonpolar solvents (cyclohexane and n -hexadecane) has been studied with ultrafast time-resolved broadband fluorescence spectroscopy. The temporal evolution of the time-resolved fluorescence spectra and bandwidth narrowing reveal a multiple-stage vibrational relaxation within ∼15 ps followed by slower excited-state deactivation steps (80 and 950 ps in cyclohexane). In addition, an oscillatory fluorescence with a period of about 1.1 ps was observed and attributed to the coherent vibrational motion of the nitrophenyl/aminophenyl torsion that modulates the early-time fluorescence spectra. The decay of the total fluorescence intensity with cubic frequency correction was found to be consistent with a competitive triplet–single deactivation mechanism.

A Molecular Nanotube with Three-Dimensional π-Conjugation.

A π-conjugated twelve-porphyrin tube is synthesized in 32 % yield by a template-directed coupling reaction that joins together six porphyrin dimers, forming twelve new C=C bonds. The nanotube has two bound templates, enclosing an internal volume of approximately 4.5 nm3. Its UV/Vis/NIR absorption and fluorescence spectra resemble those of a previously reported six-porphyrin ring, but are red-shifted by approximately 300 cm−1, reflecting increased conjugation. Ultrafast fluorescence spectroscopy demonstrates extensive excited-state delocalization. Transfer of electronic excitation from an initially formed state polarized in the direction of the nanotube axis (z axis) to an excited state polarized in the xy plane occurs within 200 fs, resulting in a negative fluorescence anisotropy on excitation at 742 nm.

A Molecular Nanotube with Three-Dimensional π-Conjugation.

A π‐conjugated twelve‐porphyrin tube is synthesized in 32 % yield by a template‐directed coupling reaction that joins together six porphyrin dimers, forming twelve new C—C bonds. The nanotube has two bound templates, enclosing an internal volume of approximately 4.5 nm3. Its UV/Vis/NIR absorption and fluorescence spectra resemble those of a previously reported six‐porphyrin ring, but are red‐shifted by approximately 300 cm−1, reflecting increased conjugation. Ultrafast fluorescence spectroscopy demonstrates extensive excited‐state delocalization. Transfer of electronic excitation from an initially formed state polarized in the direction of the nanotube axis (z axis) to an excited state polarized in the xy plane occurs within 200 fs, resulting in a negative fluorescence anisotropy on excitation at 742 nm.

A Detailed Analysis of Multiple Photoreactions in a Light-Harvesting Molecular Triad with Overlapping Spectra by Utrafast Spectroscopy

The photoreactions that occur in a molecular triad composed of derivatives of boron-dipyrromethene (BOD), diketopyrrolopyrrole (DPP), and of triphenylamine (TPA) specifically designed for the use as a possible donor material for organic solar cells are studied by ultrafast fluorescence and transient absorption spectroscopy with hyper spectral coverage (300–900 nm). While the latter is often sufficient to reveal the reaction scenario in mono- or bicomponent organic molecules, the overlap of ground state absorption and fluorescence spectra of the components present in the TPA-DPP-BOD triad requires a careful and accurate characterization of the excited state differential absorption spectra and kinetics of the individual isolated moieties. In addition, the absorption spectra of the TPA cation and the DPP and BOD anions are determined by in situ spectroelectrochemical experiments. Picosecond fluorescence demonstrates efficient excited state quenching of both DPP and BOD, with downhill energy transfer from TPA occurring in a subpicosecond time scale, leading to its complete excited state quenching. Additionally, an analysis of the multiexponential fluorescence decay indicates the existence of reactive (quenched) and nonreactive (radiative) subpopulations of DPP and BOD, most probably due to structural heterogeneities. In order to treat this complexity of multiple reaction pathways, the transient absorption data are analyzed by a combination of global fitting, providing decay-associated differential spectra (DADS), and a reconstitution of these DADS by linear combinations of the characteristic difference spectra of all molecular species and excited states. This allows us to disentangle the photoreactions that the TPA, DPP and BOD excited state subpopulations undergo on time scales ranging from 200 fs to a few ns, and to clarify the contribution of TPA as an electron donor in the formation of an intramolecular charge transfer (CT) state. DPP has a dual role as an ancillary light-harvesting unit capable of performing ultrafast energy transfer to BOD, and as an electron acceptor for the CT state. The latter has a remarkably long lifetime of 0.5 ns. We conclude that the triad could act as a very efficient electron-donor material in a blend with commonly used acceptors such as phenyl-C61-butyric acid methyl ester (PCBM).

Ultrafast Charge Transfer from CdSe Quantum Dots to p-Type NiO: Hole Injection vs Hole Trapping

Semiconductor quantum dot (QD) to metal oxide electron injection dynamics is well documented in the scientific literature. In contrast to that, not much is known so far about hole injection time scales in such systems. The current study fills this gap. We investigate photocathodes consisting of CdSe QDs and p-type NiO to study hole injection dynamics from the valence band of the QDs to NiO. The combination of two complementary techniques, ultrafast time-resolved absorption and fluorescence spectroscopies, enabled us to distinguish between hole trapping and injection. A kinetic component on the time scale of a few hundreds of picoseconds was identified as hole injection. By changing the size of the QDs, the driving force of the hole injection was tuned and we demonstrated that the hole injection rates are well described by the Marcus theory of charge transfer. In order to enhance the overall hole injection efficiency, we have passivated the CdSe QDs by a gradient ZnS shell. The core–shell QDs show signific...

Ultrafast dynamics and computational studies on diaminodicyanoquinodimethanes (DADQs).

Three diaminodicyanoquinodimethanes, 4-(R(1)R(2)C)-1-[(NC)2C]-C6H4 (R(1),R(2) = H2N, 1; R(1) = 3,5-Me2-4-OCH4H6N-, R(2) = H2N, 2; R(1) = 3,5-Me2-4-OCH4H6N-, R(2) = 4-Me-C5H9N, 3), were investigated using carbon-13 NMR, steady-state, and ultrafast transient absorption and ultrafast fluorescence spectroscopies to unravel the unusual characteristics of this class of chromophores. Computed (GIAO)B3LYP/6-31G* data for the zwitterions 1-3 using necessary solvation (PCM) models were shown to be in excellent agreement with observed structural and carbon-13 NMR data. The ground-state geometries of 1-3 contain a cationic methine group R(1)R(2)C- twisted from the C6H4 ring and an anionic methine group (NC)2C- in plane with the C6H4 ring in solution and solid state. The (13)C chemical shifts of the peak corresponding to the methine carbon at the (NC)2C- group of 1-3 are observed at 32.5-34.7 ppm, which are some 55 ppm upfield compared with the (13)C chemical shift for the methine carbons in TCNQ, 1,4-[(NC)2C]2-C6H4. The decay of the excited state in diaminodicyanoquinodimethanes is fast and dominated by nonradiative processes on the picosecond time scale, which depends on the viscosity of the medium. The dynamics of the excited-state decay is therefore limited by conformational changes through an intramolecular twisting motion. This twisting motion is hindered by friction, which, in turn, also depends on the functional group size of the system. The dominant nonradiative pathways after excitation are due to twisted excited-state conformers according to TD-DFT computations.

Implementing electrostatic polarization cannot fill the gap between experimental and theoretical measurements for the ultrafast fluorescence decay of myoglobin

Over the past few years, time-dependent ultrafast fluorescence spectroscopy method has been applied to the study of protein dynamics. However, observations from these experiments are in a controversy with other experimental studies. Participating of theoretical methods in this debate has not reconciled the contradiction, because the predicted initial relaxation from computer simulations is one-order faster than the ultrafast fluorescence spectroscopy experiment. In those simulations, pairwise force fields are employed, which have been shown to underestimate the roughness of the free energy landscape. Therefore, the relaxation rate of protein and water molecules under pairwise force fields is falsely exaggerated. In this work, we compared the relaxations of tryptophan/environment interaction under linear response approximation employing pairwise, polarized, and polarizable force fields. Results show that although the relaxation can be slowed down to a certain extent, the large gap between experiment and theory still cannot be filled.

Excited state properties of chlorophyll f in organic solvents at ambient and cryogenic temperatures

Chlorophyll f is a photosynthetic pigment that was discovered in 2010. In this study, we present investigations on spectral and dynamic characteristics of singlet-excited and triplet states of Chl f with the application of ultrafast time-resolved absorption and fluorescence spectroscopies. The pigment was studied at room temperature in two organic solvents: pyridine and diethyl ether that have different characters of coordination of the chlorophyll magnesium (Mg) atom (hexa- and penta-coordination, respectively). Cryogenic measurements (77K) were performed in 2-methyltetrahydrofuran (hexa-coordination). The singlet-excited state lifetime was measured to be 5.6ns at room temperature regardless of Mg coordination and 8.1ns at 77K. The fluorescence quantum yield of Chl f was also determined in pyridine to be 0.16. The triplet state lifetime was studied in detail in pyridine at room temperature, and the inherent lifetime was estimated to ~150μs. Selective measurements at 77K demonstrated that the metastability of the triplet state greatly enhances, and its lifetime increases by a factor of more than three.

Distinct Conformational Transition Patterns of Noncoding 7SK snRNA and HIV TAR RNAs upon Tat Binding

Noncoding 7SK snRNA is believed to play an important role in the recruitment of P-TEFb by viral protein Tat to stimulate HIV processive transcription. Because HIV-2 TAR RNA and 7SK both evolved to feature a dinucleotide bulge region, compared to the trinucleotide bulge for HIV-1 TAR, ultrafast time-resolved fluorescence spectroscopy has been used to probe the conformational landscape of HIV-2 TAR and 7SK-SL4 RNA to monitor the conformational changes upon Tat binding. Our studies demonstrate that both HIV-1/2 TAR and 7SK-SL4 sample heterogeneous ensembles in the free state and undergo distinct conformational transitions upon Tat binding. These findings provide exquisite knowledge on the conformational complexity and intricate mechanism of molecular recognition and pave the way for drug design and discovery that incorporate dynamics information.