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1.
Proc Natl Acad Sci U S A ; 113(11): 2934-9, 2016 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-26903650

RESUMO

Energy relaxation in light-harvesting complexes has been extensively studied by various ultrafast spectroscopic techniques, the fastest processes being in the sub-100-fs range. At the same time, much slower dynamics have been observed in individual complexes by single-molecule fluorescence spectroscopy (SMS). In this work, we use a pump-probe-type SMS technique to observe the ultrafast energy relaxation in single light-harvesting complexes LH2 of purple bacteria. After excitation at 800 nm, the measured relaxation time distribution of multiple complexes has a peak at 95 fs and is asymmetric, with a tail at slower relaxation times. When tuning the excitation wavelength, the distribution changes in both its shape and position. The observed behavior agrees with what is to be expected from the LH2 excited states structure. As we show by a Redfield theory calculation of the relaxation times, the distribution shape corresponds to the expected effect of Gaussian disorder of the pigment transition energies. By repeatedly measuring few individual complexes for minutes, we find that complexes sample the relaxation time distribution on a timescale of seconds. Furthermore, by comparing the distribution from a single long-lived complex with the whole ensemble, we demonstrate that, regarding the relaxation times, the ensemble can be considered ergodic. Our findings thus agree with the commonly used notion of an ensemble of identical LH2 complexes experiencing slow random fluctuations.


Assuntos
Transferência de Energia , Complexos de Proteínas Captadores de Luz/química , Espectrometria de Fluorescência/métodos , Bacterioclorofilas/química , Bacterioclorofilas/efeitos da radiação , Lasers , Luz , Complexos de Proteínas Captadores de Luz/efeitos da radiação , Microscopia Confocal , Distribuição Normal , Rodopseudomonas/química , Estatísticas não Paramétricas , Tempo
2.
J Biol Chem ; 291(32): 16730-9, 2016 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-27252376

RESUMO

The light reactions of photosynthesis, which include light-harvesting and charge separation, take place in the amphiphilic environment of the thylakoid membrane. The light-harvesting complex II (LHCII) is the main responsible for light absorption in plants and green algae and is involved in photoprotective mechanisms that regulate the amount of excited states in the membrane. The dual function of LHCII has been extensively studied in detergent micelles, but recent results have indicated that the properties of this complex differ in a lipid environment. In this work we checked these suggestions by studying LHCII in liposomes. By combining bulk and single molecule measurements, we monitored the fluorescence characteristics of liposomes containing single complexes up to densely packed proteoliposomes. We show that the natural lipid environment per se does not alter the properties of LHCII, which for single complexes remain very similar to that in detergent. However, we show that LHCII has the strong tendency to cluster in the membrane and that protein interactions and the extent of crowding modulate the lifetimes of the excited state in the membrane. Finally, the presence of LHCII monomers at low concentrations of complexes per liposome is discussed.


Assuntos
Membrana Celular/metabolismo , Chlamydomonas reinhardtii/metabolismo , Complexos de Proteínas Captadores de Luz/metabolismo , Lipídeos de Membrana/metabolismo , Membrana Celular/química , Chlamydomonas reinhardtii/química , Complexos de Proteínas Captadores de Luz/química , Lipídeos de Membrana/química
3.
Phys Chem Chem Phys ; 18(37): 25852-60, 2016 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-27604572

RESUMO

Photosystem II (PSII) is a huge pigment-protein supercomplex responsible for the primary steps of photosynthesis in green plants. Its light-harvesting antenna exhibits efficient transfer of the absorbed excitation energy to the reaction center and also contains a well-regulated protection mechanism against over-excitation in strong light conditions. The latter is based on conformational changes in antenna complexes that open up excitation decay channels resulting in considerable fluorescence quenching. Meanwhile, fluorescence blinking, observed in single antennas, is likely caused by a similar mechanism. Thus the question arises whether this effect is also present in and relevant to the native supramolecular organization of a fully assembled PSII. To further investigate energy transfer and quenching in single PSII, we performed single-molecule experiments on PSII supercomplexes at 5 °C. Analysis of the fluorescence intensity and mean lifetime allowed us to distinguish detached antennas and specifically analyze PSII supercomplexes. The average fluorescence lifetime in PSII of about 100-150 ps, measured under our extreme excitation conditions, is surprisingly similar to published ensemble lifetime data of photochemical quenching in PSII of a similar size. In our case, this lifetime is nevertheless caused by either one or multiple quenched antennas or by a quencher in the reaction center. The observed reversible light-induced changes in fluorescence intensity on a millisecond timescale are reminiscent of blinking subunits. Our results therefore directly illustrate how environmental control over a fluctuating antenna can regulate light-harvesting in plant photosynthesis.


Assuntos
Complexos de Proteínas Captadores de Luz/química , Complexo de Proteína do Fotossistema II/química , Clorofila/química , Transferência de Energia , Fluorescência , Cinética , Simulação de Dinâmica Molecular , Fotossíntese , Conformação Proteica , Multimerização Proteica , Imagem Individual de Molécula
4.
Biophys J ; 108(5): 1047-56, 2015 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-25762317

RESUMO

In the major peripheral plant light-harvesting complex LHCII, excitation energy is transferred between chlorophylls along an energetic cascade before it is transmitted further into the photosynthetic assembly to be converted into chemical energy. The efficiency of these energy transfer processes involves a complicated interplay of pigment-protein structural reorganization and protein dynamic disorder, and the system must stay robust within the fluctuating protein environment. The final, lowest energy site has been proposed to exist within a trimeric excitonically coupled chlorophyll (Chl) cluster, comprising Chls a610-a611-a612. We studied an LHCII monomer with a site-specific mutation resulting in the loss of Chls a611and a612, and find that this mutant exhibits two predominant overlapping fluorescence bands. From a combination of bulk measurements, single-molecule fluorescence characterization, and modeling, we propose the two fluorescence bands originate from differing conditions of exciton delocalization and localization realized in the mutant. Disruption of the excitonically coupled terminal emitter Chl trimer results in an increased sensitivity of the excited state energy landscape to the disorder induced by the protein conformations. Consequently, the mutant demonstrates a loss of energy transfer efficiency. On the contrary, in the wild-type complex, the strong resonance coupling and correspondingly high degree of excitation delocalization within the Chls a610-a611-a612 cluster dampens the influence of the environment and ensures optimal communication with neighboring pigments. These results indicate that the terminal emitter trimer is thus an essential design principle for maintaining the efficient light-harvesting function of LHCII in the presence of protein disorder.


Assuntos
Proteínas de Arabidopsis/metabolismo , Complexos de Proteínas Captadores de Luz/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Fluorescência , Complexos de Proteínas Captadores de Luz/química , Complexos de Proteínas Captadores de Luz/genética , Mutação , Multimerização Proteica
5.
Phys Chem Chem Phys ; 17(30): 19844-53, 2015 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-26156159

RESUMO

In light harvesting complex II (LHCII) of higher plants and green algae, carotenoids (Cars) have an important function to quench chlorophyll (Chl) triplet states and therefore avoid the production of harmful singlet oxygen. The resulting Car triplet states lead to a non-linear self-quenching mechanism called singlet-triplet (S-T) annihilation that strongly depends on the excitation density. In this work we investigated the fluorescence decay kinetics of single immobilized LHCIIs at room temperature and found a two-exponential decay with a slow (3.5 ns) and a fast (35 ps) component. The relative amplitude fraction of the fast component increases with increasing excitation intensity, and the resulting decrease in the fluorescence quantum yield suggests annihilation effects. Modulation of the excitation pattern by means of an acousto-optic modulator (AOM) furthermore allowed us to resolve the time-dependent accumulation and decay rate (∼7 µs) of the quenching species. Inspired by singlet-singlet (S-S) annihilation studies, we developed a stochastic model and then successfully applied it to describe and explain all the experimentally observed steady-state and time-dependent kinetics. That allowed us to distinctively identify the quenching mechanism as S-T annihilation. Quantitative fitting resulted in a conclusive set of parameters validating our interpretation of the experimental results. The obtained stochastic model can be generalized to describe S-T annihilation in small molecular aggregates where the equilibration time of excitations is much faster than the annihilation-free singlet excited state lifetime.


Assuntos
Complexos de Proteínas Captadores de Luz/química , Clorofila/química , Cinética , Complexos de Proteínas Captadores de Luz/metabolismo , Modelos Teóricos , Teoria Quântica , Espectrometria de Fluorescência
6.
J Phys Chem Lett ; 9(6): 1365-1371, 2018 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-29504765

RESUMO

Solar energy captured by pigments embedded in light-harvesting complexes can be transferred to neighboring pigments, dissipated, or emitted as fluorescence. Only when it reaches a reaction center is the excitation energy stabilized in the form of a charge separation and converted into chemical energy. Well-directed and regulated energy transfer within the network of pigments is therefore of crucial importance for the success of the photosynthetic processes. Using single-molecule spectroscopy, we show that phycocyanin can dynamically switch between two spectrally distinct states originating from two different conformations. Unexpectedly, one of the two states has a red-shifted emission spectrum. This state is not involved in energy dissipation; instead, we propose that it is involved in direct energy transfer to photosystem I. Finally, our findings suggest that the function of linker proteins in phycobilisomes is to stabilize one state or the other, thus controlling the light-harvesting functions of phycocyanin.

7.
J R Soc Interface ; 15(148)2018 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-30429265

RESUMO

Biological systems are dynamical, constantly exchanging energy and matter with the environment in order to maintain the non-equilibrium state synonymous with living. Developments in observational techniques have allowed us to study biological dynamics on increasingly small scales. Such studies have revealed evidence of quantum mechanical effects, which cannot be accounted for by classical physics, in a range of biological processes. Quantum biology is the study of such processes, and here we provide an outline of the current state of the field, as well as insights into future directions.


Assuntos
Biofísica/tendências , Biologia de Sistemas/tendências , Teoria Quântica
8.
Sci Rep ; 6: 26230, 2016 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-27189196

RESUMO

We derive approximate equations of motion for excited state dynamics of a multilevel open quantum system weakly interacting with light to describe fluorescence-detected single molecule spectra. Based on the Frenkel exciton theory, we construct a model for the chlorophyll part of the LHCII complex of higher plants and its interaction with previously proposed excitation quencher in the form of the lutein molecule Lut 1. The resulting description is valid over a broad range of timescales relevant for single molecule spectroscopy, i.e. from ps to minutes. Validity of these equations is demonstrated by comparing simulations of ensemble and single-molecule spectra of monomeric LHCII with experiments. Using a conformational change of the LHCII protein as a switching mechanism, the intensity and spectral time traces of individual LHCII complexes are simulated, and the experimental statistical distributions are reproduced. Based on our model, it is shown that with reasonable assumptions about its interaction with chlorophylls, Lut 1 can act as an efficient fluorescence quencher in LHCII.

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