Function of iron-stress-induced protein A in cyanobacterial cells with monomeric and trimeric photosystem I.

The acclimation of cyanobacteria to iron deficiency is crucial for their survival in natural environments. In response to iron deficiency, many cyanobacterial species induce the production of a pigment-protein complex called iron-stress-induced protein A (IsiA). IsiA proteins associate with photosystem I (PSI) and can function as light-harvesting antennas or dissipate excess energy. They may also serve as chlorophyll storage during iron limitation. In this study, we examined the functional role of IsiA in cells of Synechocystis sp. PCC 6803 grown under iron limitation conditions by measuring the cellular IsiA content and its capability to transfer energy to PSI. We specifically tested the effect of the oligomeric state of PSI by comparing wild-type (WT) Synechocystis sp. PCC 6803 with mutants lacking specific subunits of PSI, namely PsaL/PsaI (PSI subunits XI/VIII) and PsaF/PsaJ (PSI subunits III/IX). Time-resolved fluorescence spectroscopy revealed that IsiA formed functional PSI3-IsiA18 supercomplexes, wherein IsiA effectively transfers energy to PSI on a timescale of 10 ps at room temperature-measured in isolated complexes and in vivo-confirming the primary role of IsiA as an accessory light-harvesting antenna to PSI. However, a notable fraction (40%) remained unconnected to PSI, supporting the notion of a dual functional role of IsiA. Cells with monomeric PSI under iron deficiency contained, on average, only 3 to 4 IsiA complexes bound to PSI. These results show that IsiA can transfer energy to trimeric and monomeric PSI but to varying degrees and that the acclimatory production of IsiA under iron stress is controlled by its ability to perform its light-harvesting function.

Quantifying the Energy Spillover between Photosystems II and I in Cyanobacterial Thylakoid Membranes and Cells.

The spatial separation of photosystems I and II (PSI and PSII) is thought to be essential for efficient photosynthesis by maintaining a balanced flow of excitation energy between them. Unlike the thylakoid membranes of plant chloroplasts, cyanobacterial thylakoids do not form tightly appressed grana stacks that enforce strict lateral separation. The coexistence of the two photosystems provides a ground for spillover-excitation energy transfer from PSII to PSI. Spillover has been considered as a pathway of energy transfer from the phycobilisomes to PSI and may also play a role in state transitions as means to avoid overexcitation of PSII. Here, we demonstrate a significant degree of energy spillover from PSII to PSI in reconstituted membranes and isolated thylakoid membranes of Thermosynechococcus (Thermostichus) vulcanus and Synechocystis sp. PCC 6803 by steady-state and time-resolved fluorescence spectroscopy. The quantum yield of spillover in these systems was determined to be up to 40%. Spillover was also found in intact cells but to a considerably lower degree (20%) than in isolated thylakoid membranes. The findings support a model of coexistence of laterally separated microdomains of PSI and PSII in the cyanobacterial cells as well as domains where the two photosystems are energetically connected. The methodology presented here can be applied to probe spillover in other photosynthetic organisms.

Role of isotropic lipid phase in the fusion of photosystem II membranes.

It has been thoroughly documented, by using 31P-NMR spectroscopy, that plant thylakoid membranes (TMs), in addition to the bilayer (or lamellar, L) phase, contain at least two isotropic (I) lipid phases and an inverted hexagonal (HII) phase. However, our knowledge concerning the structural and functional roles of the non-bilayer phases is still rudimentary. The objective of the present study is to elucidate the origin of I phases which have been hypothesized to arise, in part, from the fusion of TMs (Garab et al. 2022 Progr Lipid Res 101,163). We take advantage of the selectivity of wheat germ lipase (WGL) in eliminating the I phases of TMs (Dlouhý et al. 2022 Cells 11: 2681), and the tendency of the so-called BBY particles, stacked photosystem II (PSII) enriched membrane pairs of 300-500 nm in diameter, to form large laterally fused sheets (Dunahay et al. 1984 BBA 764: 179). Our 31P-NMR spectroscopy data show that BBY membranes contain L and I phases. Similar to TMs, WGL selectively eliminated the I phases, which at the same time exerted no effect on the molecular organization and functional activity of PSII membranes. As revealed by sucrose-density centrifugation, magnetic linear dichroism spectroscopy and scanning electron microscopy, WGL disassembled the large laterally fused sheets. These data provide direct experimental evidence on the involvement of I phase(s) in the fusion of stacked PSII membrane pairs, and strongly suggest the role of non-bilayer lipids in the self-assembly of the TM system.

Light-adapted charge-separated state of photosystem II: structural and functional dynamics of the closed reaction center.

Photosystem II (PSII) uses solar energy to oxidize water and delivers electrons for life on Earth. The photochemical reaction center of PSII is known to possess two stationary states. In the open state (PSIIO), the absorption of a single photon triggers electron-transfer steps, which convert PSII into the charge-separated closed state (PSIIC). Here, by using steady-state and time-resolved spectroscopic techniques on Spinacia oleracea and Thermosynechococcus vulcanus preparations, we show that additional illumination gradually transforms PSIIC into a light-adapted charge-separated state (PSIIL). The PSIIC-to-PSIIL transition, observed at all temperatures between 80 and 308 K, is responsible for a large part of the variable chlorophyll-a fluorescence (Fv) and is associated with subtle, dark-reversible reorganizations in the core complexes, protein conformational changes at noncryogenic temperatures, and marked variations in the rates of photochemical and photophysical reactions. The build-up of PSIIL requires a series of light-induced events generating rapidly recombining primary radical pairs, spaced by sufficient waiting times between these events-pointing to the roles of local electric-field transients and dielectric relaxation processes. We show that the maximum fluorescence level, Fm, is associated with PSIIL rather than with PSIIC, and thus the Fv/Fm parameter cannot be equated with the quantum efficiency of PSII photochemistry. Our findings resolve the controversies and explain the peculiar features of chlorophyll-a fluorescence kinetics, a tool to monitor the functional activity and the structural-functional plasticity of PSII in different wild-types and mutant organisms and under stress conditions.

ChloroSpec: A new in vivo chlorophyll fluorescence spectrometer for simultaneous wavelength- and time-resolved detection.

Chlorophyll fluorescence is a ubiquitous tool in basic and applied plant science research. Various standard commercial instruments are available for characterization of photosynthetic material like leaves or microalgae, most of which integrate the overall fluorescence signals above a certain cut-off wavelength. However, wavelength-resolved (fluorescence signals appearing at different wavelengths having different time dependent decay) signals contain vast information required to decompose complex signals and processes into their underlying components that can untangle the photo-physiological process of photosynthesis. Hence, to address this we describe an advanced chlorophyll fluorescence spectrometer - ChloroSpec - allowing three-dimensional simultaneous detection of fluorescence intensities at different wavelengths in a time-resolved manner. We demonstrate for a variety of typical examples that most of the generally used fluorescence parameters are strongly wavelength dependent. This indicates a pronounced heterogeneity and a highly dynamic nature of the thylakoid and the photosynthetic apparatus under actinic illumination. Furthermore, we provide examples of advanced global analysis procedures integrating this three-dimensional signal and relevant information extracted from them that relate to the physiological properties of the organism. This conveniently obtained broad range of data can make ChloroSpec a new standard tool in photosynthesis research.

Trimeric photosystem I facilitates energy transfer from phycobilisomes in Synechocystis sp. PCC 6803.

In cyanobacteria, phycobilisomes (PBS) serve as peripheral light-harvesting complexes of the two photosystems, extending their antenna size and the wavelength range of photons available for photosynthesis. The abundance of PBS, the number of phycobiliproteins they contain, and their light-harvesting function are dynamically adjusted in response to the physiological conditions. PBS are also thought to be involved in state transitions that maintain the excitation balance between the two photosystems. Unlike its eukaryotic counterpart, PSI is trimeric in many cyanobacterial species and the physiological significance of this is not well understood. Here, we compared the composition and light-harvesting function of PBS in cells of Synechocystis sp. PCC 6803, which has primarily trimeric PSI, and the ΔpsaL mutant, which lacks the PsaL subunit of PSI and is unable to form trimers. We also investigated a mutant additionally lacking the PsaJ and PsaF subunits of PSI. Both strains with monomeric PSI accumulated significantly more allophycocyanin per chlorophyll, indicating higher abundance of PBS. On the other hand, a higher phycocyanin:allophycocyanin ratio in the wild type suggests larger PBS or the presence of APC-less PBS (CpcL-type) that are not assembled in cells with monomeric PSI. Steady-state and time-resolved fluorescence spectroscopy at room temperature and 77 K revealed that PSII receives more energy from the PBS at the expense of PSI in cells with monomeric PSI, regardless of the presence of PsaF. Taken together, these results show that the oligomeric state of PSI impacts the excitation energy flow in Synechocystis.

New foundations for the physical mechanism of variable chlorophyll a fluorescence. Quantum efficiency versus the light-adapted state of photosystem II.

Photosystem II (PSII) uses solar energy to oxidize water and delivers electrons to fix CO2. Although the structure at atomic resolution and the basic photophysical and photochemical functions of PSII are well understood, many important questions remain. The activity of PSII in vitro and in vivo is routinely monitored by recording the induction kinetics of chlorophyll a fluorescence (ChlF). According to the 'mainstream' model, the rise from the minimum level (Fo) to the maximum (Fm) of ChlF of dark-adapted PSII reflects the closure of all functionally active reaction centers, and the Fv/Fm ratio is equated with the maximum photochemical quantum yield of PSII (where Fv=Fm-Fo). However, this model has never been free of controversies. Recent experimental data from a number of studies have confirmed that the first single-turnover saturating flash (STSF), which generates the closed state (PSIIC), produces F1

Fluorescence quenching in thylakoid membranes induced by single-walled carbon nanotubes.

The distinct photochemical and electrochemical properties of single-walled carbon nanotubes (SWCNTs) boosted the research interest in nanomaterial utilization in different in vivo and in vitro photosynthetic biohybrid setups. Aiming to unravel the yet not fully understood energetic interactions between the nanotubes and photosynthetic pigment-protein assemblies in an aqueous milieu, we studied SWCNT effects on the photochemical reactions of isolated thylakoid membranes (TMs), Photosystem II (PSII)-enriched membrane fragments and light-harvesting complexes (LHCII). The SWCNTs induced quenching of the steady-state chlorophyll fluorescence in the TM-biohybrid systems with a corresponding shortening of the average fluorescence lifetimes. The effect was not related to changes in the integrity and macroorganization of the photosynthetic membranes. Moreover, we found no evidence for direct excitation energy exchange between the SWCNTs and pigment-protein complexes, since neither the steady-state nor time-resolved fluorescence of LHCII-biohybrid systems differed from the corresponding controls. The attenuation of the fluorescence signal in the TM-biohybrid systems indicates possible leakage of photosynthetic electrons toward the nanotubes that most probably occurs at the level of the PSII acceptor site. Although it is too early to speculate on the nature of the involved electron donors and intermediate states, the observed energetic interaction could be exploited to increase the photoelectron capture efficiency of natural biohybrid systems for solar energy conversion.

Ultrafast excitation quenching by the oxidized photosystem II reaction center.

Photosystem II (PSII) is the pigment-protein complex driving the photoinduced oxidation of water and reduction of plastoquinone in all oxygenic photosynthetic organisms. Excitations in the antenna chlorophylls are photochemically trapped in the reaction center (RC) producing the chlorophyll-pheophytin radical ion pair P+ Pheo-. When electron donation from water is inhibited, the oxidized RC chlorophyll P+ acts as an excitation quencher, but knowledge on the kinetics of quenching is limited. Here, we used femtosecond transient absorption spectroscopy to compare the excitation dynamics of PSII with neutral and oxidized RC (P+). We find that equilibration in the core antenna has a major lifetime of about 300 fs, irrespective of the RC redox state. Two-dimensional electronic spectroscopy revealed additional slower energy equilibration occurring on timescales of 3-5 ps, concurrent with excitation trapping. The kinetics of PSII with open RC can be described well with previously proposed models according to which the radical pair P+ Pheo- is populated with a main lifetime of about 40 ps, which is primarily determined by energy transfer between the core antenna and the RC chlorophylls. Yet, in PSII with oxidized RC (P+), fast excitation quenching was observed with decay lifetimes as short as 3 ps and an average decay lifetime of about 90 ps, which is shorter than the excited-state lifetime of PSII with open RC. The underlying mechanism of this extremely fast quenching prompts further investigation.

Excitation energy transfer kinetics of trimeric, monomeric and subunit-depleted Photosystem I from Synechocystis PCC 6803.

Photosystem I is the most efficient photosynthetic enzyme with structure and composition highly conserved among all oxygenic phototrophs. Cyanobacterial Photosystem I is typically associated into trimers for reasons that are still debated. Almost universally, Photosystem I contains a number of long-wavelength-absorbing 'red' chlorophylls (Chls), that have a sizeable effect on the excitation energy transfer and trapping. Here we present spectroscopic comparison of trimeric Photosystem I from Synechocystis PCC 6803 with a monomeric complex from the ΔpsaL mutant and a 'minimal' monomeric complex ΔFIJL, containing only subunits A, B, C, D, E, K and M. The quantum yield of photochemistry at room temperature was the same in all complexes, demonstrating the functional robustness of this photosystem. The monomeric complexes had a reduced far-red absorption and emission equivalent to the loss of 1.5-2 red Chls emitting at 710-715 nm, whereas the longest-wavelength emission at 722 nm was not affected. The picosecond fluorescence kinetics at 77 K showed spectrally and kinetically distinct red Chls in all complexes and equilibration times of up to 50 ps. We found that the red Chls are not irreversible traps at 77 K but can still transfer excitations to the reaction centre, especially in the trimeric complexes. Structure-based Förster energy transfer calculations support the assignment of the lowest-energy state to the Chl pair B37/B38 and the trimer-specific red Chl emission to Chls A32/B7 located at the monomer-monomer interface. These intermediate-energy red Chls facilitate energy migration from the lowest-energy states to the reaction centre.

Characterization of the Rate-Limiting Steps in the Dark-To-Light Transitions of Closed Photosystem II: Temperature Dependence and Invariance of Waiting Times during Multiple Light Reactions.

Rate-limiting steps in the dark-to-light transition of Photosystem II (PSII) were discovered by measuring the variable chlorophyll-a fluorescence transients elicited by single-turnover saturating flashes (STSFs). It was shown that in diuron-treated samples: (i) the first STSF, despite fully reducing the QA quinone acceptor molecule, generated only an F1(

Two-Dimensional Electronic Spectroscopy of a Minimal Photosystem I Complex Reveals the Rate of Primary Charge Separation.

Photosystem I (PSI), found in all oxygenic photosynthetic organisms, uses solar energy to drive electron transport with nearly 100% quantum efficiency, thanks to fast energy transfer among antenna chlorophylls and charge separation in the reaction center. There is no complete consensus regarding the kinetics of the elementary steps involved in the overall trapping, especially the rate of primary charge separation. In this work, we employed two-dimensional coherent electronic spectroscopy to follow the dynamics of energy and electron transfer in a monomeric PSI complex from Synechocystis PCC 6803, containing only subunits A-E, K, and M, at 77 K. We also determined the structure of the complex to 4.3 Å resolution by cryoelectron microscopy with refinements to 2.5 Å. We applied structure-based modeling using a combined Redfield-Förster theory to compute the excitation dynamics. The absorptive 2D electronic spectra revealed fast excitonic/vibronic relaxation on time scales of 50-100 fs from the high-energy side of the absorption spectrum. Antenna excitations were funneled within 1 ps to a small pool of chlorophylls absorbing around 687 nm, thereafter decaying with 4-20 ps lifetimes, independently of excitation wavelength. Redfield-Förster energy transfer computations showed that the kinetics is limited by transfer from these red-shifted pigments. The rate of primary charge separation, upon direct excitation of the reaction center, was determined to be 1.2-1.5 ps-1. This result implies activationless electron transfer in PSI.

Modelling excitation energy transfer and trapping in the filamentous cyanobacterium Anabaena variabilis PCC 7120.

The phycobilisome (PBS) serves as the major light-harvesting system, funnelling excitation energy to both photosystems (PS) in cyanobacteria and red algae. The picosecond kinetics involving the excitation energy transfer has been studied within the isolated systems and intact filaments of the cyanobacterium Anabaena variabilis PCC 7120. A target model is proposed which resolves the dynamics of the different chromophore groups. The energy transfer rate of 8.5 ± 1.0/ns from the rod to the core is the rate-limiting step, both in vivo and in vitro. The PBS-PSI-PSII supercomplex reveals efficient excitation energy migration from the low-energy allophycocyanin, which is the terminal emitter, in the PBS core to the chlorophyll a in the photosystems. The terminal emitter of the phycobilisome transfers energy to both PSI and PSII with a rate of 50 ± 10/ns, equally distributing the solar energy to both photosystems. Finally, the excitation energy is trapped by charge separation in the photosystems with trapping rates estimated to be 56 ± 6/ns in PSI and 14 ± 2/ns in PSII.

Characterization of fluorescent chlorophyll charge-transfer states as intermediates in the excited state quenching of light-harvesting complex II.

Light-harvesting complex II (LHCII) is the major antenna complex in higher plants and green algae. It has been suggested that a major part of the excited state energy dissipation in the so-called "non-photochemical quenching" (NPQ) is located in this antenna complex. We have performed an ultrafast kinetics study of the low-energy fluorescent states related to quenching in LHCII in both aggregated and the crystalline form. In both sample types the chlorophyll (Chl) excited states of LHCII are strongly quenched in a similar fashion. Quenching is accompanied by the appearance of new far-red (FR) fluorescence bands from energetically low-lying Chl excited states. The kinetics of quenching, its temperature dependence down to 4 K, and the properties of the FR-emitting states are very similar both in LHCII aggregates and in the crystal. No such FR-emitting states are found in unquenched trimeric LHCII. We conclude that these states represent weakly emitting Chl-Chl charge-transfer (CT) states, whose formation is part of the quenching process. Quantum chemical calculations of the lowest energy exciton and CT states, explicitly including the coupling to the specific protein environment, provide detailed insight into the chemical nature of the CT states and the mechanism of CT quenching. The experimental data combined with the results of the calculations strongly suggest that the quenching mechanism consists of a sequence of two proton-coupled electron transfer steps involving the three quenching center Chls 610/611/612. The FR-emitting CT states are reaction intermediates in this sequence. The polarity-controlled internal reprotonation of the E175/K179 aa pair is suggested as the switch controlling quenching. A unified model is proposed that is able to explain all known conditions of quenching or non-quenching of LHCII, depending on the environment without invoking any major conformational changes of the protein.

Time-resolved fluorescence study of excitation energy transfer in the cyanobacterium Anabaena PCC 7120.

Excitation energy transfer (EET) and trapping in Anabaena variabilis (PCC 7120) intact cells, isolated phycobilisomes (PBS) and photosystem I (PSI) complexes have been studied by picosecond time-resolved fluorescence spectroscopy at room temperature. Global analysis of the time-resolved fluorescence kinetics revealed two lifetimes of spectral equilibration in the isolated PBS, 30-35 ps and 110-130 ps, assigned primarily to energy transfer within the rods and between the rods and the allophycocyanin core, respectively. An additional intrinsic kinetic component with a lifetime of 500-700 ps was found, representing non-radiative decay or energy transfer in the core. Isolated tetrameric PSI complexes exhibited biexponential fluorescence decay kinetics with lifetimes of about 10 ps and 40 ps, representing equilibration between the bulk antenna chlorophylls with low-energy "red" states and trapping of the equilibrated excitations, respectively. The cascade of EET in the PBS and in PSI could be resolved in intact filaments as well. Virtually all energy absorbed by the PBS was transferred to the photosystems on a timescale of 180-190 ps.

Macroorganisation and flexibility of thylakoid membranes.

The light reactions of photosynthesis are hosted and regulated by the chloroplast thylakoid membrane (TM) - the central structural component of the photosynthetic apparatus of plants and algae. The two-dimensional and three-dimensional arrangement of the lipid-protein assemblies, aka macroorganisation, and its dynamic responses to the fluctuating physiological environment, aka flexibility, are the subject of this review. An emphasis is given on the information obtainable by spectroscopic approaches, especially circular dichroism (CD). We briefly summarise the current knowledge of the composition and three-dimensional architecture of the granal TMs in plants and the supramolecular organisation of Photosystem II and light-harvesting complex II therein. We next acquaint the non-specialist reader with the fundamentals of CD spectroscopy, recent advances such as anisotropic CD, and applications for studying the structure and macroorganisation of photosynthetic complexes and membranes. Special attention is given to the structural and functional flexibility of light-harvesting complex II in vitro as revealed by CD and fluorescence spectroscopy. We give an account of the dynamic changes in membrane macroorganisation associated with the light-adaptation of the photosynthetic apparatus and the regulation of the excitation energy flow by state transitions and non-photochemical quenching.

Redox transients of P680 associated with the incremental chlorophyll-a fluorescence yield rises elicited by a series of saturating flashes in diuron-treated photosystem II core complex of Thermosynechococcus vulcanus.

Recent chlorophyll-a fluorescence yield measurements, using single-turnover saturating flashes (STSFs), have revealed the involvement of a rate-limiting step in the reactions following the charge separation induced by the first flash. As also shown here, in diuron-inhibited PSII core complexes isolated from Thermosynechococcus vulcanus the fluorescence maximum could only be reached by a train of STSFs. In order to elucidate the origin of the fluorescence yield increments in STSF series, we performed transient absorption measurements at 819 nm, reflecting the photooxidation and re-reduction kinetics of the primary electron donor P680. Upon single flash excitation of the dark-adapted sample, the decay kinetics could be described with lifetimes of 17 ns (∼50%) and 167 ns (∼30%), and a longer-lived component (∼20%). This kinetics are attributed to re-reduction of P680•+ by the donor side of PSII. In contrast, upon second-flash (with Δt between 5 µs and 100 ms) or repetitive excitation, the 819 nm absorption changes decayed with lifetimes of about 2 ns (∼60%) and 10 ns (∼30%), attributed to recombination of the primary radical pair P680•+ Pheo•- , and a small longer-lived component (∼10%). These data confirm that only the first STSF is capable of generating stable charge separation - leading to the reduction of QA ; and thus, the fluorescence yield increments elicited by the consecutive flashes must have a different physical origin. Our double-flash experiments indicate that the rate-limiting steps, detected by chlorophyll-a fluorescence, are not correlated with the turnover of P680.

Revealing the excitation energy transfer network of Light-Harvesting Complex II by a phenomenological analysis of two-dimensional electronic spectra at 77 K.

Energy equilibration in light-harvesting antenna systems normally occurs before energy is transferred to a reaction center. The equilibration mechanism is a characteristic of the excitation energy transfer (EET) network of the antenna. Characterizing this network is crucial in understanding the first step of photosynthesis. We present our phenomenology-based analysis procedure and results in obtaining the excitonic energy levels, spectral linewidths, and transfer-rate matrix of Light-Harvesting Complex II directly from its 2D electronic spectra recorded at 77 K with waiting times between 100 fs to 100 ps. Due to the restriction of the models and complexity of the system, a unique EET network cannot be constructed. Nevertheless, a recurring pattern of energy transfer with very similar overall time scales between spectral components (excitons) is consistently obtained. The models identify a "bottleneck" state in the 664-668 nm region although with a relatively shorter lifetime (∼4-6 ps) of this state compared to previous studies. The model also determines three terminal exciton states at 675, 677-678, and 680-681 nm that are weakly coupled to each other. The excitation energy equilibration between the three termini is found to be independent of the initial excitation conditions, which is a crucial design for the light-harvesting complexes to ensure the energy flow under different light conditions and avoid excitation trapping. We proposed two EET schemes with tentative pigment assignments based on the interpretation of the modeling results together with previous structure-based calculations and spectroscopic observables.

Excitation transfer and trapping kinetics in plant photosystem I probed by two-dimensional electronic spectroscopy.

Photosystem I is a robust and highly efficient biological solar engine. Its capacity to utilize virtually every absorbed photon's energy in a photochemical reaction generates great interest in the kinetics and mechanisms of excitation energy transfer and charge separation. In this work, we have employed room-temperature coherent two-dimensional electronic spectroscopy and time-resolved fluorescence spectroscopy to follow exciton equilibration and excitation trapping in intact Photosystem I complexes as well as core complexes isolated from Pisum sativum. We performed two-dimensional electronic spectroscopy measurements with low excitation pulse energies to record excited-state kinetics free from singlet-singlet annihilation. Global lifetime analysis resolved energy transfer and trapping lifetimes closely matches the time-correlated single-photon counting data. Exciton energy equilibration in the core antenna occurred on a timescale of 0.5 ps. We further observed spectral equilibration component in the core complex with a 3-4 ps lifetime between the bulk Chl states and a state absorbing at 700 nm. Trapping in the core complex occurred with a 20 ps lifetime, which in the supercomplex split into two lifetimes, 16 ps and 67-75 ps. The experimental data could be modelled with two alternative models resulting in equally good fits-a transfer-to-trap-limited model and a trap-limited model. However, the former model is only possible if the 3-4 ps component is ascribed to equilibration with a "red" core antenna pool absorbing at 700 nm. Conversely, if these low-energy states are identified with the P700 reaction centre, the transfer-to-trap-model is ruled out in favour of a trap-limited model.

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.