Anisotropic circular dichroism signatures of oriented thylakoid membranes and lamellar aggregates of LHCII

In photosynthesis research, circular dichroism (CD) spectroscopy is an indispensable tool to probe molecular architecture at virtually all levels of structural complexity. At the molecular level, the chirality of the molecule results in intrinsic CD; pigment–pigment interactions in protein complexes and small aggregates can give rise to excitonic CD bands, while “psi-type” CD signals originate from large, densely packed chiral aggregates. It has been well established that anisotropic CD (ACD), measured on samples with defined non-random orientation relative to the propagation of the measuring beam, carries specific information on the architecture of molecules or molecular macroassemblies. However, ACD is usually combined with linear dichroism and can be distorted by instrumental imperfections, which given the strong anisotropic nature of photosynthetic membranes and complexes, might be the reason why ACD is rarely studied in photosynthesis research. In this study, we present ACD spectra, corrected for linear dichroism, of isolated intact thylakoid membranes of granal chloroplasts, washed unstacked thylakoid membranes, photosystem II (PSII) membranes (BBY particles), grana patches, and tightly stacked lamellar macroaggregates of the main light-harvesting complex of PSII (LHCII). We show that the ACD spectra of face- and edge-aligned stacked thylakoid membranes and LHCII lamellae exhibit profound differences in their psi-type CD bands. Marked differences are also seen in the excitonic CD of BBY and washed thylakoid membranes. Magnetic CD (MCD) spectra on random and aligned samples, and the largely invariable nature of the MCD spectra, despite dramatic variations in the measured isotropic and anisotropic CD, testify that ACD can be measured without substantial distortions and thus employed to extract detailed information on the (supra)molecular organization of photosynthetic complexes. An example is provided showing the ability of CD data to indicate such an organization, leading to the discovery of a novel crystalline structure in macroaggregates of LHCII.     

Atomic force microscopy of the bacterial photosynthetic apparatus: plain pictures of an elaborate machinery

Photosynthesis both in the past and present provides the vast majority of the energy used on the planet. The purple photosynthetic bacteria are a group of organisms that are able to perform photosynthesis using a particularly simple system that has been much studied. The main molecular constituents required for photosynthesis in these organisms are a small number of transmembrane pigment–protein complexes. These are able to function together with a high quantum efficiency (about 95%) to convert light energy into chemical potential energy. While the structure of the various proteins have been solved for several years, direct studies of the supramolecular assembly of these complexes in native membranes needed maturity of the atomic force microscope (AFM). Here, we review the novel findings and the direct conclusions that could be drawn from high-resolution AFM analysis of photosynthetic membranes. These conclusions rely on the possibility that the AFM brings of obtaining molecular resolution images of large membrane areas and thereby bridging the resolution gap between atomic structures and cellular ultrastructure.     

Low-temperature single-molecule spectroscopy on photosynthetic pigment–protein complexes from purple bacteria

The primary reactions of purple bacterial photosynthesis take place within two well characterized pigment–protein complexes, the core Reaction Center–Light Harvesting 1 (RC–LH1) complex and the more peripheral Light Harvesting 2 (LH2) complex. These antenna complexes serve to absorb incident solar radiation and to transfer it to the reaction-centers, where it is used to ‘power’ the photosynthetic redox reaction. This review provides an overview of how the character of the electronically excited states of these pigment–protein complexes are determined by quantum mechanics and how the respective spectral signatures can be observed by single-molecule spectroscopy.     

Organization of the pigment molecules in the chlorophyll a/b/c containing alga Mantoniella squamata (Prasinophyceae) studied by means of absorption, circular and linear dichroism spectroscopy

In order to obtain information on the organization of the pigment molecules in chlorophyll (Chl) a/b/c-containing organisms, we have carried out circular dichroism (CD), linear dichroism (LD) and absorption spectroscopic measurements on intact cells, isolated thylakoids and purified light-harvesting complexes (LHCs) of the prasinophycean alga Mantoniella squamata. The CD spectra of the intact cells and isolated thylakoids were predominated by the excitonic bands of the Chl a/b/c LHC. However, some anomalous bands indicated the existence of chiral macrodomains, which could be correlated with the multilayered membrane system in the intact cells. In the red, the thylakoid membranes and the LHC exhibited a well-discernible CD band originating from Chl c, but otherwise the CD spectra were similar to that of non-aggregated LHC II, the main Chl a/b LHC in higher plants. In the Soret region, however, an unusually intense (+) 441 nm band was observed, which was accompanied by negative bands between 465 and 510 nm. It is proposed that these bands originate from intense excitonic interactions between Chl a and carotenoid molecules. LD measurements revealed that the Q(Y) dipoles of Chl a in Mantoniella thylakoids are preferentially oriented in the plane of the membrane, with orientation angles tilting out more at shorter than at longer wavelengths (9 degrees at 677 nm, 20 degrees at 670 nm and 26 degrees at 662 nm); the Q(Y) dipole of Chl c was found to be oriented at 29 degrees with respect to the membrane plane. These data and the LD spectrum of the LHC, apart from the presence of Chl c, suggest an orientation pattern of dipoles similar to those of higher plant thylakoids and LHC II. However, the tendency of the Q(Y) dipoles of Chl b to lie preferentially in the plane of the membrane (23 degrees at 653 nm and 30 degrees at 646 nm) is markedly different from the orientation pattern in higher plant membranes and LHC II. Hence, our CD and LD data show that the molecular organization of the Chl a/b/c LHC, despite evident similarities, differs significantly from that of LHC II.     

Variation in supramolecular organisation of the photosynthetic membrane of Rhodobacter sphaeroides induced by alteration of PufX

In purple bacteria of the genus Rhodobacter (Rba.), an LH1 antenna complex surrounds the photochemical reaction centre (RC) with a PufX protein preventing the LH1 complex from completely encircling the RC. In membranes of Rba. sphaeroides, RC–LH1 complexes associate as dimers which in turn assemble into longer range ordered arrays. The present work uses linear dichroism (LD) and dark-minus-light difference LD (ΔLD) to probe the organisation of genetically altered RC–LH1 complexes in intact membranes. The data support previous proposals that Rba. capsulatus, and Rba. sphaeroides heterologously expressing the PufX protein from Rba. capsulatus, produce monomeric core complexes in membranes that lack long-range order. Similarly, Rba. sphaeroides with a point mutation in the Gly 51 residue of PufX, which is located on the membrane-periplasm interface, assembles mainly non-ordered RC–LH1 complexes that are most likely monomeric. All the Rba. sphaeroides membranes in their ΔLD spectra exhibited a spectral fingerprint of small degree of organisation implying the possibility of ordering influence of LH1, and leading to an important conclusion that PufX itself has no influence on ordering RC–LH1 complexes, as long-range order appears to be induced only through its role of configuring RC–LH1 complexes into dimers.     

Monitoring protein aggregation during thermal unfolding in circular dichroism experiments

Thermal unfolding monitored by spectroscopy or calorimetry is widely used to determine protein stability. Equilibrium thermodynamic analysis of such unfolding is often hampered by its irreversibility, which usually results from aggregation of thermally denatured protein. In addition, heat-induced protein misfolding and aggregation often lead to formation of amyloid-like structures. We propose a convenient method to monitor in real time protein aggregation during thermal folding/ unfolding transition by recording turbidity or 90 degrees light scattering data in circular dichroism (CD) spectroscopic experiments. Since the measurements of turbidity and 90 degrees light scattering can be done simultaneously with far- or near-UV CD data collection, they require no additional time or sample and can be directly correlated with the protein conformational changes monitored by CD. The results can provide useful insights into the origins of irreversible conformational changes and test the linkage between protein unfolding or misfolding and aggregation in various macromolecular systems, including globular proteins and protein-lipid complexes described in this study, as well as a wide range of amyloid-forming proteins and peptides.     

Assemblies of ionic zinc chlorins assisted by water-soluble polypeptides

Artificial chlorophyll–peptide complexes were prepared by mixing ionic chlorophyll derivatives and oligopeptides in aqueous media containing a small amount of organic solvent. The pigment–peptide complexes provided chlorophyll assemblies showing a sharp red-shifted Qy absorption band concomitant with giant circular dichroism signals. The polypeptide-assisted assemblies of chlorophyllous pigments would afford a good model for photosynthetic apparatuses such as pigment–protein complexes of light-harvesting antennas.     

Modulation of the multilamellar membrane organization and of the chiral macrodomains in the diatom Phaeodactylum tricornutum revealed by small-angle neutron scattering and circular dichroism spectroscopy

Diatoms possess effective photoprotection mechanisms, which may involve reorganizations in the photosynthetic machinery. We have shown earlier, by using circular dichroism (CD) spectroscopy, that in Phaeodactylum tricornutum the pigment-protein complexes are arranged into chiral macrodomains, which have been proposed to be associated with the multilamellar organization of the thylakoid membranes and shown to be capable of undergoing light-induced reversible reorganizations (Szabó et al. Photosynth Res 95:237, 2008). Recently, by using small-angle neutron scattering (SANS) on the same algal cells we have determined the repeat distances and revealed reversible light-induced reorganizations in the lamellar order of thylakoids (Nagy et al. Biochem J 436:225, 2011). In this study, we show that in moderately heat-treated samples, the weakening of the lamellar order is accompanied by the diminishment of the psi-type CD signal associated with the long-range chiral order of the chromophores (psi, polymer or salt-induced). Further, we show that the light-induced reversible increase in the psi-type CD is associated with swelling in the membrane system, with magnitudes larger in high light than in low light. In contrast, shrinkage of the membrane system, induced by sorbitol, brings about a decrease in the psi-type CD signal; this shrinkage also diminishes the non-photochemical quenching capability of the cells. These data shed light on the origin of the psi-type CD signal, and confirm that both CD spectroscopy and SANS provide valuable information on the macro-organization of the thylakoid membranes and their dynamic properties; these parameters are evidently of interest with regard to the photoprotection in whole algal cells.     

X-ray diffraction studies on chromatophore membrane from photosynthetic bacteria. II. Comparison of diffraction patterns of photosynthetic units from various purple bacteria

Comparative X-ray diffraction studies, in conjunction with infrared absorption spectroscopy, were performed on chromatophores isolated from various purple photosynthetic bacteria in order to achieve a better understanding of the molecular structure of the photosynthetic unit. Purple non-sulfur bacteria used were Rhodospirillum rubrum, Rhodospirillum molischianum, Rhodopseudomonas sphaeroides, and Rhodopseudomonas palustris. Chromatophores of Chromatium vinosum, as a typical example of purple sulfur bacteria, were also investigated. The results were as follows. Distinct equatorial X-ray diffraction patterns were obtained from chromatophores of all the bacteria examined. They showed diffuse, continuous diffraction patterns having several maxima, and the patterns are evidently distinguished from those of either crystalline or amorphous material. The pattern indicates that the photosynthetic unit in the chromatophore has a highly organized molecular structure in the plane of the membrane. Bacteria whose major photosynthetic pigment is bacteriochlorophyll alpha can be categorized in three groups from the viewpoint of near infrared absorption spectra. X-ray diffraction patterns are also grouped accordingly, although the differences are minimal and the patterns display common features. In other words, the bacteriochlorophyll forms, which are bacteriochlorophyll-protein complexes exhibiting different near-infrared absorption spectra, show different X-ray patterns: the molecular structure of photosynthetic units is closely related to the state of pigment in each complex, although the "X-ray" molecular structure is mainly concerned with the arrangement of constituent protein molecules at the present resolution, whereas the "spectroscopic" structure reflects the local environment of pigment.(ABSTRACT TRUNCATED AT 250 WORDS)     

An alternative model for photosystem II/light harvesting complex II in grana membranes based on cryo-electron microscopy studies.

The photosynthetic protein complexes in plants are located in the chloroplast thylakoid membranes. These membranes have an ultrastructure that consists of tightly stacked 'grana' regions interconnected by unstacked membrane regions. The structure of isolated grana membranes has been studied here by cryo-electron microscopy. The data reveals an unusual arrangement of the photosynthetic protein complexes, staggered over two tightly stacked planes. Chaotrope treatment of the paired grana membranes has allowed the separation and isolation of two biochemically distinct membrane fractions. These data have led us to an alternative model of the ultrastructure of the grana where segregation exists within the grana itself. This arrangement would change the existing view of plant photosynthesis, and suggests potential links between cyanobacterial and plant photosystem II light harvesting systems.     

The proteolysis adaptor,NblA, is essential for degradation of the core pigment of the cyanobacterial light‐harvesting complex

The cyanobacterial light‐harvesting complex, the phycobilisome, is degraded under nutrient limitation, allowing the cell to adjust light absorbance to its metabolic capacity. This large light‐harvesting antenna comprises a core complex of the pigment allophycocyanin, and rod‐shaped pigment assemblies emanating from the core. NblA, a low‐molecular‐weight protein, is essential for degradation of the phycobilisome. NblA mutants exhibit high absorbance of rod pigments under conditions that generally elicit phycobilisome degradation, implicating NblA in degradation of these pigments. However, the vast abundance of rod pigments and the substantial overlap between the absorbance spectra of rod and core pigments has made it difficult to directly associate NblA with proteolysis of the phycobilisome core. Furthermore, lack of allophycocyanin degradation in an NblA mutant may reflect a requirement for rod degradation preceding core degradation, and does not prove direct involvement of NblA in proteolysis of the core pigment. Therefore, in this study, we used a mutant lacking phycocyanin, the rod pigment of Synechococcus elongatusPCC7942, to examine whether NblA is required for allophycocyanin degradation. We demonstrate that NblA is essential for degradation of the core complex of the phycobilisome. Furthermore, fluorescence lifetime imaging microscopy provided in situ evidence for the interaction of NblA with allophycocyanin, and indicated that NblA interacts with allophycocyanin complexes that are associated with the photosynthetic membranes. Based on these data, as well as previous observations indicating interaction of NblA with phycobilisomes attached to the photosynthetic membranes, we suggest a model for sequential phycobilisome disassembly by NblA.     

Organization in photosynthetic membranes of purple bacteria in vivo: The role of carotenoids

Photosynthesis in purple bacteria is performed by pigment–protein complexes that are closely packed within specialized intracytoplasmic membranes. Here we report on the influence of carotenoid composition on the organization of RC–LH1 pigment–protein complexes in intact membranes and cells of Rhodobacter sphaeroides. Mostly dimeric RC–LH1 complexes could be isolated from strains expressing native brown carotenoids when grown under illuminated/anaerobic conditions, or from strains expressing green carotenoids when grown under either illuminated/anaerobic or dark/semiaerobic conditions. However, mostly monomeric RC–LH1 complexes were isolated from strains expressing the native photoprotective red carotenoid spheroidenone, which is synthesized during phototrophic growth in the presence of oxygen. Despite this marked difference, linear dichroism (LD) and light-minus-dark LD spectra of oriented intact intracytoplasmic membranes indicated that RC–LH1 complexes are always assembled in ordered arrays, irrespective of variations in the relative amounts of isolated dimeric and monomeric RC–LH1 complexes. We propose that part of the photoprotective response to the presence of oxygen mediated by synthesis of spheroidenone may be a switch of the structure of the RC–LH1 complex from dimers to monomers, but that these monomers are still organized into the photosynthetic membrane in ordered arrays. When levels of the dimeric RC–LH1 complex were very high, and in the absence of LH2, LD and ?LD spectra from intact cells indicated an ordered arrangement of RC–LH1 complexes. Such a degree of ordering implies the presence of highly elongated, tubular membranes with dimensions requiring orientation along the length of the cell and in a proportion larger than previously observed.     

Low pH induced structural reorganization in thylakoid membranes

By using low temperature fluorescence spectroscopy, it has been shown that exposing chloroplast thylakoid membranes to acidic pH reversibly decreases the fluorescence of photosystem II while the fluorescence of photosystem I increases [P. Singh-Rawal et al. (2010) Evidence that pH can drive state transitions in isolated thylakoid membranes from spinach, Photochem Photobiol Sci, 9 830-837]. In order to shed light on the origin of these changes, we performed circular dichroism (CD) spectroscopy on freshly isolated pea thylakoid membranes. We show that the magnitude of the psi-type CD, which is associated with the presence of chirally ordered macroarrays of the chromophores in intact thylakoid membranes, decreases gradually and reversibly upon gradually lowering the pH of the medium from 7.5 to 4.5 (psi, polymer or salt induced). The same treatment, as shown on thylakoid membranes washed in hypotonic low salt medium possessing no psi-type bands, induces no discernible change in the excitonic CD. These data show that while no change in the pigment-pigment interactions and thus in the molecular organization of the bulk protein complexes can be held responsible for the observed changes in the fluorescence, acidification of the medium significantly alters the macro-organization of the complexes, hence providing an explanation for the pH-induced redistribution of the excitation energy between the two photosystems. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Linear-dichroism spectroscopy for the study of structural properties of proteins

This review gives an experiment directed survey of the application of linear-dichroism (LD) spectroscopy to the study of proteins. LD spectroscopy is a relatively simple technique that provides information on the orientation of chromophores in molecules, on molecular characteristies such as shape, size and electronic properties, and on binding parameters in molecular complexes. Since LD is only observed when the molecules are non-randomly oriented in the sample, particular attention is paid to various orientation techniques, viz. in electric and flow fields, in polymer films and gels, and by light induction (photoselection). Examples are given on bacteriorhodopsin and retinals, chlorosomes, lens crystallins, aspartate aminotransferase, and the interaction of gene32-and recA-protein with DNA.Abbreviations AAT aspartate aminotransferase - BR bacteriorhodopsin - CD circular dichroism - LD linear dichroism - PLP pyridoxal-5-phosphate - ss single stranded - TDM transition dipole moment     

Parameterization of an effective potential for protein–ligand binding from host–guest affinity data

Force field accuracy is still one of the “stalemates” in biomolecular modeling. Model systems with high quality experimental data are valuable instruments for the validation and improvement of effective potentials. With respect to protein–ligand binding, organic host–guest complexes have long served as models for both experimental and computational studies because of the abundance of binding affinity data available for such systems. Binding affinity data collected for cyclodextrin (CD) inclusion complexes, a popular model for molecular recognition, is potentially a more reliable resource for tuning energy parameters than hydration free energy measurements. Convergence of binding free energy calculations on CD host–guest systems can also be obtained rapidly, thus offering the opportunity to assess the robustness of these parameters. In this work, we demonstrate how implicit solvent parameters can be developed using binding affinity experimental data and the binding energy distribution analysis method (BEDAM) and validated using the Grid Inhomogeneous Solvation Theory analysis. These new solvation parameters were used to study protein–ligand binding in two drug targets against the HIV‐1 virus and improved the agreement between the calculated and the experimental binding affinities. This work illustrates how benchmark sets of high quality experimental binding affinity data and physics‐based binding free energy models can be used to evaluate and optimize force fields for protein–ligand systems. Copyright © 2015 John Wiley & Sons, Ltd.     

Circular dichroism of films of polynucleotides

The CD spectra of films of the lithium salt of E. coli and calf thymus DNA, and alternating d-AT : AT were measured as a function of relative humidity. Films of the ammonium acetate salt of DNA were also measured. The ammonium films yield the previously reported A-form CD spectra. A possible explanation for the small magnitude of the 260-nm band of the A-form film spectra compared to double-stranded RNA spectra is that the film DNA is in a different conformation than RNA within the A family of conformations. At relative humidities of 92% or lower, a negative nonconservative CD spectrum with negative minima near 270 and 210 nm is observed with the lithium films. The magnitude of the minima varies from film to film. In films of DNA the magnitude ranges from a delta epsilon of ?5 to ?35; d-AT : AT films show magnitudes to ?300. CD spectra of this type are designated Ψ spectra. Similar spectra have been reported from reconstituted complexes of DNA and polylysine or f-1 histone. If the origins of the film and protein–DNA complex spectra are similar, the complex spectra are not the result of specific secondary structural changes induced in the DNA by the protein fraction. Theoretical analysis suggests that Ψ spectra are not the result of changes in the secondary or tertiary structure of DNA. Instead, the previously proposed explanation based on liquid crystals is favored. The DNA could form asymmetric structures with long-range periodicity. It is likely that the observed CD spectra of f-1 complexes are artifacts of DNA aggregation. The possibility that some other previously published spectra of protein–DNA complexes also reflect artifacts is suggested.     

Importance of trimer-trimer interactions for the native state of the plant light-harvesting complex II

Aggregates and solubilized trimers of LHCII were characterized by circular dichroism (CD), linear dichroism and time-resolved fluorescence spectroscopy and compared with thylakoid membranes in order to evaluate the native state of LHCII in vivo. It was found that the CD spectra of lamellar aggregates closely resemble those of unstacked thylakoid membranes whereas the spectra of trimers solubilized in n-dodecyl-beta,D-maltoside, n-octyl-beta,D-glucopyranoside, or Triton X-100 were drastically different in the Soret region. Thylakoid membranes or LHCII aggregates solubilized with detergent exhibited CD spectra similar to the isolated trimers. Solubilization of LHCII was accompanied by profound changes in the linear dichroism and increase in fluorescence lifetime. These data support the notion that lamellar aggregates of LHCII retain the native organization of LHCII in the thylakoid membranes. The results indicate that the supramolecular organization of LHCII, most likely due to specific trimer-trimer contacts, has significant impact on the pigment interactions in the complexes.