Excited-state dynamics
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Fluorescence methods are widely used in studies of biological and model membranes. The dynamics of membrane fluorescent markers in their ground and excited electronic states and correlations with their molecular surrounding within the fully hydrated phospholipid bilayer are still not well understood. In the present work, Quantum Mechanical (QM) calculations and Molecular Dynamics (MD) simulations are used to characterize location and interactions of two membrane polarity probes (Prodan; 6-propionyl-2-dimethylaminonaphthalene and its derivative Laurdan; 2-dimethylamino-6-lauroylnaphthalene) with the dioleoylphosphatidylcholine (DOPC) lipid bilayer model. MD simulations with fluorophores in ground and excited states are found to be a useful tool to analyze the fluorescent dye dynamics and their immediate vicinity. The results of QM calculations and MD simulations are in excellent agreement with available experimental data. The calculation shows that the two amphiphilic dyes initially placed in bulk water diffuse within 10 ns towards their final location in the lipid bilayer. Analysis of solvent relaxation process in the aqueous phase occurs on the picoseconds timescale whereas it takes nanoseconds at the lipid/water interface. Four different relaxation time constants, corresponding to different relaxation processes, where observed when the dyes were embedded into the membrane.
Light-harvesting complex II (LHCII) from the marine green macroalga Bryopsis corticulans is spectroscopically characterized to understand the structural and functional changes resulting from adaptation to intertidal environment. LHCII is homologous to its counterpart in land plants but has a different carotenoid and chlorophyll (Chl) composition. This is reflected in the steady-state absorption, fluorescence, linear dichroism, circular dichroism and anisotropic circular dichroism spectra. Time-resolved fluorescence and two-dimensional electronic spectroscopy were used to investigate the consequences of this adaptive change in the pigment composition on the excited-state dynamics. The complex contains additional Chl b spectral forms - absorbing at around 650 nm and 658 nm - and lacks the red-most Chl a forms compared with higher-plant LHCII. Similar to plant LHCII, energy transfer between Chls occurs on timescales from under hundred fs (mainly from Chl b to Chl a) to several picoseconds (mainly between Chl a pools). However, the presence of long-lived, weakly coupled Chl b and Chl a states leads to slower exciton equilibration in LHCII from B. corticulans. The finding demonstrates a trade-off between the enhanced absorption of blue-green light and the excitation migration time. However, the adaptive change does not result in a significant drop in the overall photochemical efficiency of Photosystem II. These results show that LHCII is a robust adaptable system whose spectral properties can be tuned to the environment for optimal light harvesting.
Photosynthetic eukaryotes whose cells harbor plastids originating from secondary endosymbiosis of a red alga include species of major ecological and economic importance. Since utilization of solar energy relies on the efficient light-harvesting, one of the critical factors for the success of the red lineage in a range of environments is to be found in the adaptability of the light-harvesting machinery, formed by the proteins of the light-harvesting complex (LHC) family. A number of species are known to employ mainly a unique class of LHC containing red-shifted chlorophyll a (Chl a) forms absorbing above 690 nm. This appears to be an adaptation to shaded habitats. Here we present a detailed investigation of excitation energy flow in the red-shifted light-harvesting antenna of eustigmatophyte Trachydiscus minutus using time-resolved fluorescence and ultrafast transient absorption measurements. The main carotenoid in the complex is violaxanthin, hence this LHC is labeled the red-violaxanthin-Chl a protein, rVCP. Both the carotenoid-to-Chl a energy transfer and excitation dynamics within the Chl a manifold were studied and compared to the related antenna complex, VCP, that lacks the red-Chl a. Two spectrally defined carotenoid pools were identified in the red antenna, contributing to energy transfer to Chl a, mostly via S2 and hot S1 states. Also, Chl a triplet quenching by carotenoids is documented. Two separate pools of red-shifted Chl a were resolved, one is likely formed by excitonically coupled Chl a molecules. The structural implications of these observations are discussed.
- MeSH
- chlorofyl a * MeSH
- Chlorophyta fyziologie MeSH
- fluorescenční spektrometrie metody MeSH
- Heterokontophyta fyziologie MeSH
- plastidy MeSH
- přenos energie fyziologie MeSH
- Rhodophyta fyziologie MeSH
- světlosběrné proteinové komplexy chemie MeSH
- xanthofyly MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In this study, the vibrational characteristics of optically excited echinenone in various solvents and the Orange Carotenoid Protein (OCP) in red and orange states are systematically investigated through steady-state and time-resolved spectroscopy techniques. Time-resolved experiments, employing both Transient Absorption (TA) and Femtosecond Stimulated Raman Spectroscopy (FSRS), reveal different states in the OCP photoactivation process. The time-resolved studies indicate vibrational signatures of exited states positioned above the S1 state during the initial 140 fs of carotenoid evolution in OCP, an absence of a vibrational signature for the relaxed S1 state of echinenone in OCP, and more robust signatures of a highly excited ground state (GS) in OCP. Differences in S1 state vibration population signatures between OCP and solvents are attributed to distinct conformations of echinenone in OCP and hydrogen bonds at the keto group forming a short-lived intramolecular charge transfer (ICT) state. The vibrational dynamics of the hot GS in OCP show a more pronounced red shift of ground state CC vibration compared to echinenone in solvents, thus suggesting an unusually hot form of GS. The study proposes a hypothesis for the photoactivation mechanism of OCP, emphasizing the high level of vibrational excitation in longitudinal stretching modes as a driving force. In conclusion, the comparison of vibrational signatures reveals unique dynamics of energy dissipation in OCP, providing insights into the photoactivation mechanism and highlighting the impact of the protein environment on carotenoid behavior. The study underscores the importance of vibrational analysis in understanding the intricate processes involved in early phase OCP photoactivation.
The activity of the light-oxygen-voltage/helix-turn-helix (LOV-HTH) photoreceptor EL222 is regulated through protein-protein and protein-DNA interactions, both triggered by photo-excitation of its flavin mononucleotide (FMN) cofactor. To gain molecular-level insight into the photocycle of EL222, we applied complementary methods: macromolecular X-ray crystallography (MX), nuclear magnetic resonance (NMR) spectroscopy, optical spectroscopies (infrared and UV-visible), molecular dynamics/metadynamics (MD/metaD) simulations, and protein engineering using noncanonical amino acids. Kinetic experiments provided evidence for two distinct EL222 conformations (lit1 and lit2) that become sequentially populated under illumination. These two lit states were assigned to covalently bound N5 protonated, and noncovalently bound hydroquinone forms of FMN, respectively. Only subtle structural differences were observed between the monomeric forms of all three EL222 species (dark, lit1, and lit2). While the dark state is largely monomeric, both lit states undergo monomer-dimer exchange. Furthermore, molecular modeling revealed differential dynamics and interdomain separation times arising from the three FMN states (oxidized, adduct, and reduced). Unexpectedly, all three EL222 species can associate with DNA, but only upon blue-light irradiation, a high population of stable complexes is obtained. Overall, we propose a model of EL222 activation where photoinduced changes in the FMN moiety shift the population equilibrium toward an open conformation that favors self-association and DNA-binding.
- MeSH
- bakteriální proteiny chemie metabolismus MeSH
- DNA vazebné proteiny chemie metabolismus MeSH
- DNA * chemie metabolismus MeSH
- flavinmononukleotid * chemie metabolismus MeSH
- flaviny chemie metabolismus MeSH
- kinetika MeSH
- konformace proteinů MeSH
- krystalografie rentgenová MeSH
- oxidace-redukce * MeSH
- simulace molekulární dynamiky MeSH
- světlo * MeSH
- Thermosynechococcus metabolismus MeSH
- transkripční faktory metabolismus chemie MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
Coherent vibration states in biological systems excited in nonlinear electrically polar structures by metabolic energy supply were postulated by H. Frohlich. Frohlich's requirements for coherent vibrations and generation of electromagnetic field are satisfied by microtubules whose subunits are electric dipoles. Static electric field around mitochondria and "wasted energy" efflux from them provide nonlinear conditions and coherent excitation. Numerical models are used for analysis of coherent vibration states. A hypothesis is presented that dysfunction of mitochondria (i.e., extinction of the zones of the static electric field and of the efflux of "wasted energy") and disintegration of the cytoskeleton on the pathway of cancer transformation result in disturbances of coherence of the cellular electrically polar oscillations and of the generated electromagnetic field with consequences in cellular organization and interactions between cells. Local invasion, detachment, and metastasis of cancer cells are subsequent events of disturbed electromagnetic interactions.
- MeSH
- biofyzikální jevy MeSH
- biologické modely MeSH
- cytoskelet metabolismus účinky záření MeSH
- elektromagnetická pole škodlivé účinky MeSH
- lidé MeSH
- mitochondrie metabolismus účinky záření MeSH
- nádorová transformace buněk MeSH
- nádorové buněčné linie MeSH
- nádory patologie MeSH
- nelineární dynamika MeSH
- vibrace MeSH
- Check Tag
- lidé MeSH
The fucoxanthin-chlorophyll a protein from Emiliania huxleyi (E-FCP) is a member of the LHC family of light-harvesting proteins. It has a rather unusual pigment composition as its binds more Chl-c than Chl-a, and 19'-hexanoyloxyfucoxanthin (hFx) as the main carotenoid instead of fucoxanthin (Fx) typically found in various FCP complexes. The presence of a hexanoyloxy tail in hFx suppresses the charge transfer character of the S1/ICT state resulting in almost no effect of polarity on the excited state dynamics of hFx, strongly contrasting with the excited-state properties of Fx. Here we report on the dynamics of the energy transfer between hFx and Chl in E-FCP, and we compare it with Fx-Chl energy transfer in the FCP complex from Phaeodactylum tricornutum. In both complexes, the excited hFx (Fx) transfers energy from the S2 state with a sub-100 fs time constant and no effect of the hexanoyloxy tail on the efficiency of the S2 route was found. The energy transfer via the S1/ICT state has in E-FCP two channels characterized by 1.5 and 11 ps time constants, while for FCP these two channels operate with time constants of 0.8 and 4.5 ps. Thus, minimizing the charge transfer character of S1/ICT in hFx results in about twice slower energy transfer via the S1/ICT state, underlining the importance of the ICT state in facilitating carotenoid-Chl energy transfer in systems utilizing keto carotenoids as energy donors.
The keto-carotenoid deinoxanthin, which occurs in the UV-resistant bacterium Deinococcus radiodurans, has been investigated by ultrafast time-resolved spectroscopy techniques. We have explored the excited-state properties of deinoxanthin in solution and bound to the S-layer Deinoxanthin Binding Complex (SDBC), a protein complex important for UV resistance and thermostability of the organism. Binding of deinoxanthin to SDBC shifts the absorption spectrum to longer wavelengths, but excited-state dynamics remain unaffected. The lifetime of the lowest excited state (S1) of isolated deinoxanthin in methanol is 2.1 ps. When bound to SDBC, the S1 lifetime is 2.4 ps, indicating essentially no alteration of the effective conjugation length upon binding. Moreover, our data show that the conformational disorder in both ground and excited states is the same for deinoxanthin in methanol and bound to SDBC. Our results thus suggest a rather loosely bound carotenoid in SDBC, making it very distinct from other carotenoid-binding proteins such as Orange Carotenoid Protein (OCP) or crustacyanin, both of which significantly restrain the carotenoid at the binding site. Three deinoxanthin analogs were found to bind the SDBC, suggesting a non-selective binding site of deinoxanthin in SDBC.
The major light-harvesting complex of Amphidinium (A.) carterae, chlorophyll-a-chlorophyll-c 2-peridinin-protein complex (acpPC), was studied using ultrafast pump-probe spectroscopy at low temperature (60 K). An efficient peridinin-chlorophyll-a energy transfer was observed. The stimulated emission signal monitored in the near-infrared spectral region was stronger when redder part of peridinin pool was excited, indicating that these peridinins have the S1/ICT (intramolecular charge-transfer) state with significant charge-transfer character. This may lead to enhanced energy transfer efficiency from "red" peridinins to chlorophyll-a. Contrary to the water-soluble antenna of A. carterae, peridinin-chlorophyll-a protein, the energy transfer rates in acpPC were slower under low-temperature conditions. This fact underscores the influence of the protein environment on the excited-state dynamics of pigments and/or the specificity of organization of the two pigment-protein complexes.
- MeSH
- blízká infračervená spektroskopie * MeSH
- časové faktory MeSH
- chlorofyl metabolismus MeSH
- Dinoflagellata metabolismus MeSH
- elektrony MeSH
- karotenoidy metabolismus MeSH
- kinetika MeSH
- nízká teplota * MeSH
- přenos energie MeSH
- světlosběrné proteinové komplexy metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Chl synthase (ChlG) is an important enzyme of the Chl biosynthetic pathway catalyzing attachment of phytol/geranylgeraniol tail to the chlorophyllide molecule. Here we have investigated the Flag-tagged ChlG (f.ChlG) in a complex with two different high-light inducible proteins (Hlips) HliD and HliC. The f.ChlG-Hlips complex binds a Chl a and three different carotenoids, β-carotene, zeaxanthin and myxoxanthophyll. Application of ultrafast time-resolved absorption spectroscopy performed at room and cryogenic temperatures revealed excited-state dynamics of complex-bound pigments. After excitation of Chl a in the complex, excited Chl a is efficiently quenched by a nearby carotenoid molecule via energy transfer from the Chl a Qy state to the carotenoid S1 state. The kinetic analysis of the spectroscopic data revealed that quenching occurs with a time constant of ~2ps and its efficiency is temperature independent. Even though due to its long conjugation myxoxanthophyll appears to be energetically best suited for role of Chl a quencher, based on comparative analysis and spectroscopic data we propose that β-carotene bound to Hlips acts as the quencher rather than myxoxanthophyll and zeaxanthin, which are bound at the f.ChlG and Hlips interface. The S1 state lifetime of the quencher has been determined to be 13ps at room temperature and 21ps at 77K. These results demonstrate that Hlips act as a conserved functional module that prevents photodamage of protein complexes during photosystem assembly or Chl biosynthesis.
- MeSH
- bakteriální proteiny chemie MeSH
- fotolýza MeSH
- karotenoidy farmakologie MeSH
- ligasy tvořící vazby C-O chemie MeSH
- sinice enzymologie MeSH
- světlosběrné proteinové komplexy chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
