Cryo-EM structures of light-harvesting 2 complexes from Rhodopseudomonas palustris reveal the molecular origin of absorption tuning.

The genomes of some purple photosynthetic bacteria contain a multigene puc family encoding a series of α- and ß-polypeptides that together form a heterogeneous antenna of light-harvesting 2 (LH2) complexes. To unravel this complexity, we generated four sets of puc deletion mutants in Rhodopseudomonas palustris, each encoding a single type of pucBA gene pair and enabling the purification of complexes designated as PucA-LH2, PucB-LH2, PucD-LH2, and PucE-LH2. The structures of all four purified LH2 complexes were determined by cryogenic electron microscopy (cryo-EM) at resolutions ranging from 2.7 to 3.6 Å. Uniquely, each of these complexes contains a hitherto unknown polypeptide, γ, that forms an extended undulating ribbon that lies in the plane of the membrane and that encloses six of the nine LH2 αß-subunits. The γ-subunit, which is located near to the cytoplasmic side of the complex, breaks the C9 symmetry of the LH2 complex and binds six extra bacteriochlorophylls (BChls) that enhance the 800-nm absorption of each complex. The structures show that all four complexes have two complete rings of BChls, conferring absorption bands centered at 800 and 850 nm on the PucA-LH2, PucB-LH2, and PucE-LH2 complexes, but, unusually, the PucD-LH2 antenna has only a single strong near-infared (NIR) absorption peak at 803 nm. Comparison of the cryo-EM structures of these LH2 complexes reveals altered patterns of hydrogen bonds between LH2 αß-side chains and the bacteriochlorin rings, further emphasizing the major role that H bonds play in spectral tuning of bacterial antenna complexes.

Reproducibility and accuracy of microscale thermophoresis in the NanoTemper Monolith: a multi laboratory benchmark study.

Microscale thermophoresis (MST), and the closely related Temperature Related Intensity Change (TRIC), are synonyms for a recently developed measurement technique in the field of biophysics to quantify biomolecular interactions, using the (capillary-based) NanoTemper Monolith and (multiwell plate-based) Dianthus instruments. Although this technique has been extensively used within the scientific community due to its low sample consumption, ease of use, and ubiquitous applicability, MST/TRIC has not enjoyed the unambiguous acceptance from biophysicists afforded to other biophysical techniques like isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR). This might be attributed to several facts, e.g., that various (not fully understood) effects are contributing to the signal, that the technique is licensed to only a single instrument developer, NanoTemper Technology, and that its reliability and reproducibility have never been tested independently and systematically. Thus, a working group of ARBRE-MOBIEU has set up a benchmark study on MST/TRIC to assess this technique as a method to characterize biomolecular interactions. Here we present the results of this study involving 32 scientific groups within Europe and two groups from the US, carrying out experiments on 40 Monolith instruments, employing a standard operation procedure and centrally prepared samples. A protein-small molecule interaction, a newly developed protein-protein interaction system and a pure dye were used as test systems. We characterized the instrument properties and evaluated instrument performance, reproducibility, the effect of different analysis tools, the influence of the experimenter during data analysis, and thus the overall reliability of this method.

Carotenoid Nuclear Reorganization and Interplay of Bright and Dark Excited States.

We report quantum chemical calculations using multireference perturbation theory (MRPT) with the density matrix renormalization group (DMRG) plus photothermal deflection spectroscopy measurements to investigate the manifold of carotenoid excited states and establish their energies relative to the bright state (S2) as a function of nuclear reorganization. We conclude that the primary photophysics and function of carotenoids are determined by interplay of only the bright (S2) and lowest-energy dark (S1) states. The lowest-lying dark state, far from being energetically distinguishable from the lowest-lying bright state along the entire excited-state nuclear reorganization pathway, is instead computed to be either the second or first excited state depending on what equilibrium geometry is considered. This result suggests that, rather than there being a dark intermediate excited state bridging a non-negligible energy gap from the lowest-lying dark state to the lowest-lying bright state, there is in fact no appreciable energy gap to bridge following photoexcitation. Instead, excited-state nuclear reorganization constitutes the bridge from S2 to S1, in the sense that these two states attain energetic degeneracy along this pathway.

Characterisation of a pucBA deletion mutant from Rhodopseudomonas palustris lacking all but the pucBAd genes.

Rhodopseudomonas palustris is a species of purple photosynthetic bacteria that has a multigene family of puc genes that encode the alpha and beta apoproteins, which form the LH2 complexes. A genetic dissection strategy has been adopted in order to try and understand which spectroscopic form of LH2 these different genes produce. This paper presents a characterisation of one of the deletion mutants generated in this program, the pucBAd only mutant. This mutant produces an unusual spectroscopic form of LH2 that only has a single large NIR absorption band at 800 nm. Spectroscopic and pigment analyses on this complex suggest that it has basically a similar overall structure as that of the wild-type HL LH2 complex. The mutant has the unique phenotype where the mutant LH2 complex is only produced when cells are grown at LL. At HL the mutant only produces the LH1-RC core complex.

Vibronic coupling in the excited-states of carotenoids.

The ultrafast femtochemistry of carotenoids is governed by the interaction between electronic excited states, which has been explained by the relaxation dynamics within a few hundred femtoseconds from the lowest optically allowed excited state S2 to the optically dark state S1. Extending this picture, some additional dark states (3A(g)(-) and 1B(u)(-)) and their interaction with the S2 state have also been suggested to play a major role in the ultrafast deactivation of carotenoids and their properties. Here, we investigate the interaction between such dark and bright electronic excited states of open chain carotenoids, particularly its dependence on the number of conjugated double bonds (N). We focus on the ultrafast wave packet motion on the excited potential surface, which is modified by the interaction between bright and dark electronic states. Such a coupling between electronic states leads to a shift of the vibrational frequency during the excited-state evolution. In this regard, pump-degenerate four-wave mixing (pump-DFWM) is applied to a series of carotenoids with different numbers of conjugated double bonds N = 9, 10, 11 and 13 (neurosporene, spheroidene, lycopene and spirilloxanthin, respectively). Moreover, we demonstrate in a closed-chain carotenoid (lutein) that the coupling strength and therefore the vibrational shift can be tailored by changing the energy degeneracy between the 1B(u)(+) and 1B(u)(-) states via solvent interaction.

Dark States in the Light-Harvesting complex 2 Revealed by Two-dimensional Electronic Spectroscopy.

Energy transfer and trapping in the light harvesting antennae of purple photosynthetic bacteria is an ultrafast process, which occurs with a quantum efficiency close to unity. However the mechanisms behind this process have not yet been fully understood. Recently it was proposed that low-lying energy dark states, such as charge transfer states and polaron pairs, play an important role in the dynamics and directionality of energy transfer. However, it is difficult to directly detect those states because of their small transition dipole moment and overlap with the B850/B870 exciton bands. Here we present a new experimental approach, which combines the selectivity of two-dimensional electronic spectroscopy with the availability of genetically modified light harvesting complexes, to reveal the presence of those dark states in both the genetically modified and the wild-type light harvesting 2 complexes of Rhodopseudomonas palustris. We suggest that Nature has used the unavoidable charge transfer processes that occur when LH pigments are concentrated to enhance and direct the flow of energy.

Fluorescence polarization measures energy funneling in single light-harvesting antennas--LH2 vs conjugated polymers.

Numerous approaches have been proposed to mimic natural photosynthesis using artificial antenna systems, such as conjugated polymers (CPs), dendrimers, and J-aggregates. As a result, there is a need to characterize and compare the excitation energy transfer (EET) properties of various natural and artificial antennas. Here we experimentally show that EET in single antennas can be characterized by 2D polarization imaging using the single funnel approximation. This methodology addresses the ability of an individual antenna to transfer its absorbed energy towards a single pool of emissive states, using a single parameter called energy funneling efficiency (ε). We studied individual peripheral antennas of purple bacteria (LH2) and single CP chains of 20 nm length. As expected from a perfect antenna, LH2s showed funneling efficiencies close to unity. In contrast, CPs showed lower average funneling efficiencies, greatly varying from molecule to molecule. Cyclodextrin insulation of the conjugated backbone improves EET, increasing the fraction of CPs possessing ε = 1. Comparison between LH2s and CPs shows the importance of the protection systems and the protein scaffold of LH2, which keep the chromophores in functional form and at such geometrical arrangement that ensures excellent EET.

Multi-Level, Multi Time-Scale Fluorescence Intermittency of Photosynthetic LH2 Complexes: A Precursor of Non-Photochemical Quenching?

The light harvesting complex LH2 is a chromoprotein that is an ideal system for studying protein dynamics via the spectral fluctuations of the emission of its intrinsic chromophores. We have immobilized these complexes in a polymer film and studied the fluctuations of the fluorescence intensity from individual complexes over 9 orders of magnitude in time. Combining time-tagged detection of single photons with a change-point analysis has allowed the unambigeous identification of the various intensity levels due to the huge statistical basis of the data set. We propose that the observed intensity level fluctuations reflect conformational changes of the protein backbone that might be a precursor of the mechanism from which nonphotochemical quenching of higher plants has evolved.

Conformational Memory of a Protein Revealed by Single-Molecule Spectroscopy.

Proteins are supramolecular machines that carry out a wide range of different functions, many of which require flexibility. Up until now spontaneous conformational fluctuations of proteins have always been assumed to reflect a stochastic random process. However, if changing between different conformational states was random, then it would be difficult to understand how conformational control of protein function could have evolved. Here we demonstrate that a single protein can show conformational memory. This is exactly the process that can facilitate the evolution of control of switching between two conformational states that can then be used to regulate protein function.

The open, the closed, and the empty: time-resolved fluorescence spectroscopy and computational analysis of RC-LH1 complexes from Rhodopseudomonas palustris.

We studied the time-resolved fluorescence of isolated RC-LH1 complexes from Rhodopseudomonas palustris as a function of the photon fluence and the repetition rate of the excitation laser. Both parameters were varied systematically over 3 orders of magnitude. On the basis of a microstate description we developed a quantitative model for RC-LH1 and obtained very good agreement between experiments and elaborate simulations based on a global master equation approach. The model allows us to predict the relative population of RC-LH1 complexes with the special pair in the neutral state or in the oxidized state P(+) and those complexes that lack a reaction center.

Single-molecule spectroscopy unmasks the lowest exciton state of the B850 assembly in LH2 from Rps. acidophila.

We have recorded fluorescence-excitation and emission spectra from single LH2 complexes from Rhodopseudomonas (Rps.) acidophila. Both types of spectra show strong temporal spectral fluctuations that can be visualized as spectral diffusion plots. Comparison of the excitation and emission spectra reveals that for most of the complexes the lowest exciton transition is not observable in the excitation spectra due to the cutoff of the detection filter characteristics. However, from the spectral diffusion plots we have the full spectral and temporal information at hand and can select those complexes for which the excitation spectra are complete. Correlating the red most spectral feature of the excitation spectrum with the blue most spectral feature of the emission spectrum allows an unambiguous assignment of the lowest exciton state. Hence, application of fluorescence-excitation and emission spectroscopy on the same individual LH2 complex allows us to decipher spectral subtleties that are usually hidden in traditional ensemble spectroscopy.

Does the reconstitution of RC-LH1 complexes from Rhodopseudomonas acidophila strain 10050 into a phospholipid bilayer yield the optimum environment for optical spectroscopy?

We have investigated reaction-center light-harvesting 1 (RC-LH1) complexes from Rhodopseudomonas (Rps.) acidophila in detergent buffer solution and reconstituted into a phospholipid bilayer and compared the results with the outcome of an earlier study conducted on RC-LH1 immobilized in polyvinyl alcohol (PVA). The aim of this study was to test whether the immobilization of the complexes in a PVA matrix might lead to a deterioration of the proteins and thereby limit the accessible information that can be obtained from optical spectroscopy. It has been found that the complexes dissolved in a detergent buffer solution are subject to fast spectral dynamics preventing any meaningful application of single-molecule spectroscopy. In contrast, for the bilayer samples it is revealed that the reconstitution process results in a significantly larger fraction of broken complexes with respect to the preparation of the complexes in a PVA film. Moreover, we find that for the intact complexes the statistics of the key spectral features, such as the spectral separations of the bands and the mutual orientation of their transition-dipole moments, show no variation dependent on using either a bilayer or PVA as a matrix. Given the additional effort involved in the reconstitution process, the lower amount of intact RC-LH1 complexes and, concerning the decisive spectral details, the identical results with respect to embedding the complexes in a PVA matrix, we come to the conclusion that the immobilization of these proteins in a PVA matrix is a good choice for conducting low-temperature experiments on individual light-harvesting complexes.

Fluorescence-excitation and emission spectra from LH2 antenna complexes of Rhodopseudomonas acidophila as a function of the sample preparation conditions.

The high sensitivity of optical spectra of pigment-protein complexes to temperature and pressure is well known. In the present study, we have demonstrated the significant influence of the environments commonly used in bulk and single-molecule spectroscopic studies at low temperatures on the LH2 photosynthetic antenna complex from Rhodopseudomonas acidophila. A transfer of this LH2 complex from a bulk-buffer solution into a spin-coated polymer film results in a 189 cm(-1) blue shift of the B850 excitonic absorption band at 5 K. Within the molecular exciton model, the origin of this shift could be disentangled into three parts, namely to an increase of the local site energies, a contraction of the exciton band, and a decrease of the displacement energy.

Highlighting short-lived excited electronic states with pump-degenerate-four-wave-mixing.

Detection of short-lived transient species is a major challenge in femtosecond spectroscopy, especially when third-order techniques like transient absorption are used. Higher order methods employ additional interactions between light and matter to highlight such transient species. In this work we address numerically and experimentally the detection of ultrafast species with pump-Degenerate Four Wave Mixing (pump-DFWM). In this respect, conclusive identification of ultrafast species requires the proper determination of time-zero between all four laser pulses (pump pulse and the DFWM sequence). This is addressed here under the light of experimental parameters as well as molecular properties: The role of pulse durations, amount of pulse chirp as well as excited state life time is investigated by measuring a row of natural pigments differing mainly in the number of conjugated double bonds (N = 9 to 13). A comparison of the different signals reveals a strikingly unusual behavior of spheroidene (N = 10). Complete analysis of the pump-DFWM signal illustrates the power of the method and clearly assigns the uniqueness of spheroidene to a mixing of the initially excited state with a dark excited electronic state.

Single-molecule spectroscopy reveals photosynthetic LH2 complexes switch between emissive states.

Photosynthetic organisms flourish under low light intensities by converting photoenergy to chemical energy with near unity quantum efficiency and under high light intensities by safely dissipating excess photoenergy and deleterious photoproducts. The molecular mechanisms balancing these two functions remain incompletely described. One critical barrier to characterizing the mechanisms responsible for these processes is that they occur within proteins whose excited-state properties vary drastically among individual proteins and even within a single protein over time. In ensemble measurements, these excited-state properties appear only as the average value. To overcome this averaging, we investigate the purple bacterial antenna protein light harvesting complex 2 (LH2) from Rhodopseudomonas acidophila at the single-protein level. We use a room-temperature, single-molecule technique, the anti-Brownian electrokinetic trap, to study LH2 in a solution-phase (nonperturbative) environment. By performing simultaneous measurements of fluorescence intensity, lifetime, and spectra of single LH2 complexes, we identify three distinct states and observe transitions occurring among them on a timescale of seconds. Our results reveal that LH2 complexes undergo photoactivated switching to a quenched state, likely by a conformational change, and thermally revert to the ground state. This is a previously unobserved, reversible quenching pathway, and is one mechanism through which photosynthetic organisms can adapt to changes in light intensities.

Single-molecule spectroscopy on RC-LH1 complexes of Rhodopseudomonas acidophila strain 10050.

We have revisited the RC-LH1 complex from Rhodopseudomonas (Rps.) acidophila for single-molecule spectroscopy. For the current study the pigment-protein complexes were stabilized in the detergent buffer solution using a relatively mild detergent (dodecyl-ß-D-maltoside (DDM) instead of lauryldimethylamine N-oxide (LDAO)). This leads to a significant reduction of the fraction of broken/dissociated RC-LH1 complexes with respect to previous studies and has allowed us to investigate a sufficiently large sample of individual RC-LH1 complexes. For most of the complexes the fluorescence-excitation spectra exhibit a narrow spectral feature at the red end of the spectrum. Analysis of the statistics of the spectral properties yields a close resemblance with the results obtained on RC-LH1 complexes from Rps. palustris for which a low-resolution X-ray structure is available. Based on this comparison we come to the conclusion that for both species the RC-LH1 complex can be described by the same structural model, that is, an overall elliptical assembly of pigments that features a gap.

Exciton self trapping in photosynthetic pigment-protein complexes studied by single-molecule spectroscopy.

Evidence for the formation of self-trapped exciton states in photosynthetic antenna complexes is provided by comparing single-molecule fluorescence-excitation and emission spectra that have been recorded from the same individual LH2 complex from Rhodopseudomonas acidophila . While the excitation spectra showed the signatures for the B800 and B850 bands as observed previously, two distinctively different types of emission spectra were found. One group of antenna complexes shows spectra with a relatively narrow spectral profile with a clear signature of a zero-phonon line, whereas the other group of complexes displays spectra that consist only of a broad featureless band. Analysis of these data reveals clear correlations between the spectral position of the emission, the width of the spectral profile, and the associated electron-phonon coupling strength.

The electronically excited states of LH2 complexes from Rhodopseudomonas acidophila strain 10050 studied by time-resolved spectroscopy and dynamic Monte Carlo simulations. II. Homo-arrays of LH2 complexes reconstituted into phospholipid model membranes.

We performed time-resolved spectroscopy on homoarrays of LH2 complexes from the photosynthetic purple bacterium Rhodopseudomonas acidophila. Variations of the fluorescence transients were monitored as a function of the excitation fluence and the repetition rate of the excitation. These parameters are directly related to the excitation density within the array and to the number of LH2 complexes that still carry a triplet state prior to the next excitation. Comparison of the experimental observations with results from dynamic Monte Carlo simulations for a model cluster of LH2 complexes yields qualitative agreement without the need for any free parameter and reveals the mutual relationship between energy transfer and annihilation processes.