Aggregation-related quenching of LHCII fluorescence in liposomes revealed by single-molecule spectroscopy.

Incorporation of membrane proteins into reconstituted lipid membranes is a common approach for studying their structure and function relationship in a native-like environment. In this work, we investigated fluorescence properties of liposome-reconstituted major light-harvesting complexes of plants (LHCII). By utilizing liposome labelling with the fluorescent dye molecules and single-molecule microscopy techniques, we were able to study truly liposome-reconstituted LHCII and compare them with bulk measurements and liposome-free LHCII aggregates bound to the surface. Our results showed that fluorescence lifetime obtained in bulk and in single liposome measurements were correlated. The fluorescence lifetimes of LHCII were shorter for liposome-free LHCII than for reconstituted LHCII. In the case of liposome-reconstituted LHCII, fluorescence lifetime showed dependence on the protein density reminiscent to concentration quenching. The dependence of fluorescence lifetime of LHCII on the liposome size was not significant. Our results demonstrated that fluorescence quenching can be induced by LHCII - LHCII interactions in reconstituted membranes, most likely occurring via the same mechanism as photoprotective non-photochemical quenching in vivo.

Fluorescence Microscopy of Single Liposomes with Incorporated Pigment-Proteins.

Reconstitution of transmembrane proteins into liposomes is a widely used method to study their behavior under conditions closely resembling the natural ones. However, this approach does not allow precise control of the liposome size, reconstitution efficiency, and the actual protein-to-lipid ratio in the formed proteoliposomes, which might be critical for some applications and/or interpretation of data acquired during the spectroscopic measurements. Here, we present a novel strategy employing methods of proteoliposome preparation, fluorescent labeling, purification, and surface immobilization that allow us to quantify these properties using fluorescence microscopy at the single-liposome level and for the first time apply it to study photosynthetic pigment-protein complexes LHCII. We show that LHCII proteoliposome samples, even after purification with a density gradient, always contain a fraction of nonreconstituted protein and are extremely heterogeneous in both protein density and liposome sizes. This strategy enables quantitative analysis of the reconstitution efficiency of different protocols and precise fluorescence spectroscopic study of various transmembrane proteins in a controlled nativelike environment.

Dynamic feedback of the photosystem II reaction centre on photoprotection in plants.

Photosystem II of higher plants is protected against light damage by thermal dissipation of excess excitation energy, a process that can be monitored through non-photochemical quenching of chlorophyll fluorescence. When the light intensity is lowered, non-photochemical quenching largely disappears on a time scale ranging from tens of seconds to many minutes. With the use of picosecond fluorescence spectroscopy, we demonstrate that one of the underlying mechanisms is only functional when the reaction centre of photosystem II is closed, that is when electron transfer is blocked and the risk of photodamage is high. This is accompanied by the appearance of a long-wavelength fluorescence band. As soon as the reaction centre reopens, this quenching, together with the long-wavelength fluorescence, disappears instantaneously. This allows plants to maintain a high level of photosynthetic efficiency even in dangerous high-light conditions.

Is There Excitation Energy Transfer between Different Layers of Stacked Photosystem-II-Containing Thylakoid Membranes?

We have compared picosecond fluorescence decay kinetics for stacked and unstacked photosystem II membranes in order to evaluate the efficiency of excitation energy transfer between the neighboring layers. The measured kinetics were analyzed in terms of a recently developed fluctuating antenna model that provides information about the dimensionality of the studied system. Independently of the stacking state, all preparations exhibited virtually the same value of the apparent dimensionality, d = 1.6. Thus, we conclude that membrane stacking does not affect the efficiency of the delivery of excitation energy toward the reaction centers but ensures a more compact organization of the thylakoid membranes within the chloroplast and separation of photosystems I and II.

Excitation migration in fluctuating light-harvesting antenna systems.

Complex multi-exponential fluorescence decay kinetics observed in various photosynthetic systems like photosystem II (PSII) have often been explained by the reversible quenching mechanism of the charge separation taking place in the reaction center (RC) of PSII. However, this description does not account for the intrinsic dynamic disorder of the light-harvesting proteins as well as their fluctuating dislocations within the antenna, which also facilitate the repair of RCs, state transitions, and the process of non-photochemical quenching. Since dynamic fluctuations result in varying connectivity between pigment-protein complexes, they can also lead to non-exponential excitation decay kinetics. Based on this presumption, we have recently proposed a simple conceptual model describing excitation diffusion in a continuous medium and accounting for possible variations of the excitation transfer pathways. In the current work, this model is further developed and then applied to describe fluorescence kinetics originating from very diverse antenna systems, ranging from PSII of various sizes to LHCII aggregates and even the entire thylakoid membrane. In all cases, complex multi-exponential fluorescence kinetics are perfectly reproduced on the entire relevant time scale without assuming any radical pair equilibration at the side of the excitation quencher, but using just a few parameters reflecting the mean excitation energy transfer rate as well as the overall average organization of the photosynthetic antenna.

Light harvesting in a fluctuating antenna.

One of the major players in oxygenic photosynthesis, photosystem II (PSII), exhibits complex multiexponential fluorescence decay kinetics that for decades has been ascribed to reversible charge separation taking place in the reaction center (RC). However, in this description the protein dynamics is not taken into consideration. The intrinsic dynamic disorder of the light-harvesting proteins along with their fluctuating dislocations within the antenna inevitably result in varying connectivity between pigment-protein complexes and therefore can also lead to nonexponential excitation decay kinetics. On the basis of this presumption, we propose a simple conceptual model describing excitation diffusion in a continuous medium and accounting for possible variations of the excitation transfer rates. Recently observed fluorescence kinetics of PSII of different sizes are perfectly reproduced with only two adjustable parameters instead of the many decay times and amplitudes required in standard analysis procedures; no charge recombination in the RC is required. The model is also able to provide valuable information about the structural and functional organization of the photosynthetic antenna and in a straightforward way solves various contradictions currently existing in the literature.

Exciton band structure in bacterial peripheral light-harvesting complexes.

The variability of the exciton spectra of bacteriochlorophyll molecules in light-harvesting (LH) complexes of photosynthetic bacteria ensures the excitation energy funneling trend toward the reaction center. The decisive shift of the energies is achieved due to exciton spectra formation caused by the resonance interaction between the pigments. The possibility to resolve the upper Davydov sub-band corresponding to the B850 ring and, thus, to estimate the exciton bandwidth by analyzing the temperature dependence of the steady-state absorption spectra of the LH2 complexes is demonstrated. For this purpose a self-modeling curve resolution approach was applied for analysis of the temperature dependence of the absorption spectra of LH2 complexes from the photosynthetic bacteria Rhodobacter (Rba.) sphaeroides and Rhodoblastus (Rbl.) acidophilus. Estimations of the intradimer resonance interaction values as follows directly from obtained estimations of the exciton bandwidths at room temperature give 385 and 397 cm(-1) for the LH2 complexes from the photosynthetic bacteria Rba. sphaeroides and Rhl. acidophilus, respectively. At 4 K the corresponding couplings are slightly higher (391 and 435 cm(-1), respectively). The retained exciton bandwidth at physiological conditions supports the decisive role of the exciton coherence determining light absorption in bacterial light-harvesting antenna complexes.

Excitation migration, quenching, and regulation of photosynthetic light harvesting in photosystem II.

Excitation energy transfer and quenching in LHCII aggregates is considered in terms of a coarse-grained model. The model assumes that the excitation energy transfer within a pigment-protein complex is much faster than the intercomplex excitation energy transfer, whereas the quenching ability is attributed to a specific pigment-protein complex responsible for the nonphotochemical quenching (NPQ). It is demonstrated that the pump-probe experimental data obtained at low excitation intensities for LHCII aggregates under NPQ conditions can be equally well explained at two limiting cases, either describing the excitation kinetics in the migration-limited or in the trap-limited regime. Thus, it is concluded that low excitation conditions do not allow one to unambiguously define the relationship between the mean times of excitation migration and trapping. However, this could be achieved by using high excitation conditions when exciton-exciton annihilation is dominant. In this case it was found that in the trap-limited regime the excitation kinetics in the aggregate should be almost insensitive to the excitation density, meaning that singlet-singlet annihilation has little effect on the NPQ decay kinetics, whereas in the migration-limited case there is a clear intensity dependence. In order to account for the random distribution of the NPQ-traps within the LHCII aggregates, excitation diffusion in a continuous medium with random static traps was considered. This description demonstrates a very good correspondence to the experimental fluorescence kinetics assuming a lamellar (quasi-3D) structure of the antenna characterized by the dimension d=2.4 and therefore justifying the diffusion-limited approach on which the model is based. Using the coarse-grained model to describe the aggregate we estimate one NPQ-trap per 100 monomeric LHCII complexes. Finally we discuss the origin of the traps responsible for excitation quenching under NPQ conditions.

Davydov splitting of excitons in cyclic bacteriochlorophyll a nanoaggregates of bacterial light-harvesting complexes between 4.5 and 263 K.

The nature of electronic excitations created by photon absorption in the cyclic B850 aggregates of 18 bacteriochlorophyll molecules of LH2 antenna complexes of photosynthetic bacteria is studied over a broad temperature range using absorption, fluorescence, and fluorescence anisotropy spectra. The latter technique has been proved to be suitable for revealing the hidden structure of excitons in inhomogeneously broadened spectra of cyclic aggregates. A theoretical model that accounts for differences of absorbing excitons in undeformed and emitting exciton polarons in deformed antenna lattices is also developed. Only a slight decrease of the exciton bandwidth and exciton coupling energy with temperature is observed. Survival of excitons in the whole temperature span from cryogenic to nearly ambient temperatures strongly suggests that collective, coherent electronic excitations might play a role in the functional light-harvesting process taking place at physiological temperatures.

Effect of antenna-depletion in Photosystem II on excitation energy transfer in Arabidopsis thaliana.

The role of individual photosynthetic antenna complexes of Photosystem II (PSII) both in membrane organization and excitation energy transfer have been investigated. Thylakoid membranes from wild-type Arabidopsis thaliana, and three mutants lacking light-harvesting complexes CP24, CP26, or CP29, respectively, were studied by picosecond-fluorescence spectroscopy. By using different excitation/detection wavelength combinations it was possible for the first time, to our knowledge, to separate PSI and PSII fluorescence kinetics. The sub-100 ps component, previously ascribed entirely to PSI, turns out to be due partly to PSII. Moreover, the migration time of excitations from antenna to PSII reaction center (RC) was determined for the first time, to our knowledge, for thylakoid membranes. It is four times longer than for PSII-only membranes, due to additional antenna complexes, which are less well connected to the RC. The results in the absence of CP26 are very similar to those of wild-type, demonstrating that the PSII organization is not disturbed. However, the kinetics in the absence of CP29 and, especially, of CP24 show that a large fraction of the light-harvesting complexes becomes badly connected to the RCs. Interestingly, the excited-state lifetimes of the disconnected light-harvesting complexes seem to be substantially quenched.

Modeling of exciton quenching in photosystem II.

Excitation energy transfer and trapping by the artificially postulated traps in photosystem II (PSII) were modeled in terms of a coarse-grained model. The model is based on the assumption that the excitation energy transfer within a pigment-protein complex is much faster than the intercomplex excitation energy transfer. As a result, the excitation energy transfer and trapping rates by the reaction center (RC) were rescaled by the relevant entropic factors and an additional trapping rate for a specific pigment-protein complex responsible for the non-photochemical quenching (NPQ) had to be included into the theoretical framework. For the analysis, dimeric models of PSII were considered. The efficiency of the excitation quenching by the NPQ traps, depending on their positioning and on the trapping rate, was analyzed. It was concluded that the highest efficiency of the NPQ quencher could be achieved when they are localized in the major light-harvesting complexes, LHCII, and the excitation relaxation in this state is fast, of the order of picoseconds and even faster. The origin of the state responsible for NPQ is discussed.

Determination of the excitation migration time in Photosystem II consequences for the membrane organization and charge separation parameters.

The fluorescence decay kinetics of Photosystem II (PSII) membranes from spinach with open reaction centers (RCs), were compared after exciting at 420 and 484 nm. These wavelengths lead to preferential excitation of chlorophyll (Chl) a and Chl b, respectively, which causes different initial excited-state populations in the inner and outer antenna system. The non-exponential fluorescence decay appears to be 4.3+/-1.8 ps slower upon 484 nm excitation for preparations that contain on average 2.45 LHCII (light-harvesting complex II) trimers per reaction center. Using a recently introduced coarse-grained model it can be concluded that the average migration time of an electronic excitation towards the RC contributes approximately 23% to the overall average trapping time. The migration time appears to be approximately two times faster than expected based on previous ultrafast transient absorption and fluorescence measurements. It is concluded that excitation energy transfer in PSII follows specific energy transfer pathways that require an optimized organization of the antenna complexes with respect to each other. Within the context of the coarse-grained model it can be calculated that the rate of primary charge separation of the RC is (5.5+/-0.4 ps)(-1), the rate of secondary charge separation is (137+/-5 ps)(-1) and the drop in free energy upon primary charge separation is 826+/-30 cm(-1). These parameters are in rather good agreement with recently published results on isolated core complexes [Y. Miloslavina, M. Szczepaniak, M.G. Muller, J. Sander, M. Nowaczyk, M. Rögner, A.R. Holzwarth, Charge separation kinetics in intact Photosystem II core particles is trap-limited. A picosecond fluorescence study, Biochemistry 45 (2006) 2436-2442].

Excitation energy transfer and charge separation in photosystem II membranes revisited.

We have performed time-resolved fluorescence measurements on photosystem II (PSII) containing membranes (BBY particles) from spinach with open reaction centers. The decay kinetics can be fitted with two main decay components with an average decay time of 150 ps. Comparison with recent kinetic exciton annihilation data on the major light-harvesting complex of PSII (LHCII) suggests that excitation diffusion within the antenna contributes significantly to the overall charge separation time in PSII, which disagrees with previously proposed trap-limited models. To establish to which extent excitation diffusion contributes to the overall charge separation time, we propose a simple coarse-grained method, based on the supramolecular organization of PSII and LHCII in grana membranes, to model the energy migration and charge separation processes in PSII simultaneously in a transparent way. All simulations have in common that the charge separation is fast and nearly irreversible, corresponding to a significant drop in free energy upon primary charge separation, and that in PSII membranes energy migration imposes a larger kinetic barrier for the overall process than primary charge separation.

Selective recruitment of membrane protein complexes onto gold substrates patterned by dip-pen nanolithography.

Dip-pen nanolithography (DPN) is employed to develop a generic array platform for the selective recruitment of membrane protein complexes. An atomic force microscope tip inked with HS(CH2)16NH2 is used to generate amino-terminated domains on gold. These domains can be arranged into microscopic and submicroscopic patterns, and the untreated gold substrate is subsequently blocked with HS(CH2)2CONH(CH2CH2O)15CH3, a compound known to resist the unspecific binding of proteins and cells. The patterned gold substrate is exposed to an enriched membrane fraction from mutant Rhodobacter sphaeroides, which contains photosynthetic core complexes consisting of the reaction center and the light-harvesting complex LH1. The selective recruitment to the patterned domains, governed primarily by electrostatic interactions, is confirmed by contact mode atomic force microscopy.

Red chlorophylls in the exciton model of photosystem I.

Structural arrangement of pigment molecules of Photosystem I of photosynthetic cyanobacterium Synechococcus elongatus is used for theoretical modeling of the excitation energy spectrum. It is demonstrated that a straightforward application of the exciton theory with the assumption of the same molecular transition energy does not describe the red side of the absorption spectrum. Since the inhomogeneity in the molecular transition energies caused by a dispersive interaction with the molecular surrounding cannot be identified directly from the structural model, the evolutionary search procedure is used for fitting the low temperature absorption and circular dichroism spectra. As a result, one dimer, three trimers and one tetramer of chlorophyll molecules responsible for the red side of the absorption spectrum with their assignment to the spectroscopically established three bands at 708, 714 and 719 nm are determined. All of them are found to be situated not in the very close vicinity of the reaction center but are encircling it almost at the same distance. In order to explain the unusual broadening on the red side of the spectrum the exciton state mixing with the charge transfer (CT) states is considered. It is shown that two effects can be distinguished as caused by mixing of those states: (i) the oscillator strength borrowing by the CT state from the exciton transition and (ii) the borrowing of the high density of the CT state by the exciton state. The intermolecular vibrations between two counter-charged molecules determine the high density in the CT state. From the broad red absorption wing it is concluded that the CT state should be the lowest state in the complexes under consideration. Such mixing effect enables resolving the diversity in the molecular transition energies as determined by different theoretical approaches.