PRE-driven protein NMR structures: an alternative approach in highly paramagnetic systems.

Metalloproteins play key roles across biology, and knowledge of their structure is essential to understand their physiological role. For those metalloproteins containing paramagnetic states, the enhanced relaxation caused by the unpaired electrons often makes signal detection unfeasible near the metal center, precluding adequate structural characterization right where it is more biochemically relevant. Here, we report a protein structure determination by NMR where two different sets of restraints, one containing Nuclear Overhauser Enhancements (NOEs) and another containing Paramagnetic Relaxation Enhancements (PREs), are used separately and eventually together. The protein PioC from Rhodopseudomonas palustris TIE-1 is a High Potential Iron-Sulfur Protein (HiPIP) where the [4Fe-4S] cluster is paramagnetic in both oxidation states at room temperature providing the source of PREs used as alternative distance restraints. Comparison of the family of structures obtained using NOEs only, PREs only, and the combination of both reveals that the pairwise root-mean-square deviation (RMSD) between them is similar and comparable with the precision within each family. This demonstrates that, under favorable conditions in terms of protein size and paramagnetic effects, PREs can efficiently complement and eventually replace NOEs for the structural characterization of small paramagnetic metalloproteins and de novo-designed metalloproteins by NMR. DATABASES: The 20 conformers with the lowest target function constituting the final family obtained using the full set of NMR restraints were deposited to the Protein Data Bank (PDB ID: 6XYV). The 20 conformers with the lowest target function obtained using NOEs only (PDB ID: 7A58) and PREs only (PDB ID: 7A4L) were also deposited to the Protein Data Bank. The chemical shift assignments were deposited to the BMRB (code 34487).

Combinatory actions of CP29 phosphorylation by STN7 and stability regulate leaf age-dependent disassembly of photosynthetic complexes.

A predominant physiological change that occurs during leaf senescence is a decrease in photosynthetic efficiency. An optimal organization of photosynthesis complexes in plant leaves is critical for efficient photosynthesis. However, molecular mechanisms for regulating photosynthesis complexes during leaf senescence remain largely unknown. Here we tracked photosynthesis complexes alterations during leaf senescence in Arabidopsis thaliana. Grana stack is significantly thickened and photosynthesis complexes were disassembled in senescing leaves. Defects in STN7 and CP29 led to an altered chloroplast ultrastructure and a malformation of photosynthesis complex organization in stroma lamella. Both CP29 phosphorylation by STN7 and CP29 fragmentation are highly associated with the photosynthesis complex disassembly. In turn, CP29 functions as a molecular glue to facilitate protein complex formation leading phosphorylation cascade and to maintain photosynthetic efficiency during leaf senescence. These data suggest a novel molecular mechanism to modulate leaf senescence via CP29 phosphorylation and fragmentation, serving as an efficient strategy to control photosynthesis complexes.

Structure of a symmetric photosynthetic reaction center-photosystem.

Reaction centers are pigment-protein complexes that drive photosynthesis by converting light into chemical energy. It is believed that they arose once from a homodimeric protein. The symmetry of a homodimer is broken in heterodimeric reaction-center structures, such as those reported previously. The 2.2-angstrom resolution x-ray structure of the homodimeric reaction center-photosystem from the phototroph Heliobacterium modesticaldum exhibits perfect C2 symmetry. The core polypeptide dimer and two small subunits coordinate 54 bacteriochlorophylls and 2 carotenoids that capture and transfer energy to the electron transfer chain at the center, which performs charge separation and consists of 6 (bacterio)chlorophylls and an iron-sulfur cluster; unlike other reaction centers, it lacks a bound quinone. This structure preserves characteristics of the ancestral reaction center, providing insight into the evolution of photosynthesis.

Ultra-high-resolution structure and charge-density analysis of high-potential iron-sulfur protein.

The high-potential iron-sulfur protein (HiPIP) is a small (~ 80 residues) soluble metalloprotein functioning as an electron carrier in photosynthetic bacteria. HiPIP has one Fe4 S4 cluster at its molecular center. Its electronic structure is important for understanding electron transport. We recently succeeded in determining an ultra-high-resolution structure and analyzing the charge-density of HiPIP by using X-ray diffraction data at 0.48 Å resolution. The distribution of valence electrons in the iron-sulfur cluster and in the protein environment were clearly visualized, which is the first successful case for metalloproteins. In addition, a topological analysis of the charge density provided information about the electronic structure of the cluster.

Towards structural and functional characterization of photosynthetic and mitochondrial supercomplexes.

Bioenergetic reactions in chloroplasts and mitochondria are catalyzed by large multi-subunit membrane proteins. About two decades ago it became clear that several of these large membrane proteins further associate into supercomplexes and since then a number of new ones have been described. In this review we focus on supercomplexes involved in light harvesting and electron transfer in the primary reactions of oxygenic photosynthesis and on the mitochondrial supercomplexes that catalyze electron transfer and ATP synthesis in oxidative phosphorylation. Functional and structural aspects are overviewed. In addition, several relevant technical aspects are discussed, including membrane solubilization with suitable detergents and methods of purification. Some open questions are addressed, such as the lack of high-resolution structures, the outstanding gaps in the knowledge about supercomplexes involved in cyclic electron transport in photosynthesis and the unusual mitochondrial protein complexes of protists and in particular of ciliates.

Structure and binding efficiency relations of QB site inhibitors of photosynthetic reaction centres.

Many herbicides employed in agriculture and also some antibiotics bind to a specific site of the reaction centre protein (RC) blocking the photosynthetic electron transport. Crystal structures showed that all these compounds bind at the secondary ubiquinone (QB) site albeit to slightly different places. Different herbicide molecules have different binding affinities (evaluated as inhibition constants, KI, and binding enthalpy values, ΔHbind). The action of inhibitors depends on the following parameters: (i) herbicide molecular structure; (ii) interactions between herbicide and quinone binding site; (iii) protein environment. In our investigations KI and ΔHbind were determined for several inhibitors. Bound herbicide structures were optimized and their intramolecular charge distributions were calculated. Experimental and calculated data were compared to those available from databank crystal structures. We can state that the herbicide inhibition efficiency depends on steric and electronic, i.e. geometry of binding with the protein and molecular charge distribution, respectively. Apolar bulky groups on N-7 atom of the inhibitor molecule (like t-buthyl in terbutryn) are preferable for establishing stronger interactions with QB site, while such substituents are not recommended on N-8. The N-4,7,8 nitrogen atoms maintain a larger electron density so that more effective H-bonds are formed between the inhibitor and the surrounding amino acids of the protein.

Structure and energy transfer in photosystems of oxygenic photosynthesis.

Oxygenic photosynthesis is the principal converter of sunlight into chemical energy on Earth. Cyanobacteria and plants provide the oxygen, food, fuel, fibers, and platform chemicals for life on Earth. The conversion of solar energy into chemical energy is catalyzed by two multisubunit membrane protein complexes, photosystem I (PSI) and photosystem II (PSII). Light is absorbed by the pigment cofactors, and excitation energy is transferred among the antennae pigments and converted into chemical energy at very high efficiency. Oxygenic photosynthesis has existed for more than three billion years, during which its molecular machinery was perfected to minimize wasteful reactions. Light excitation transfer and singlet trapping won over fluorescence, radiation-less decay, and triplet formation. Photosynthetic reaction centers operate in organisms ranging from bacteria to higher plants. They are all evolutionarily linked. The crystal structure determination of photosynthetic protein complexes sheds light on the various partial reactions and explains how they are protected against wasteful pathways and why their function is robust. This review discusses the efficiency of photosynthetic solar energy conversion.

Supramolecular organization of photosynthetic membrane proteins in the chlorosome-containing bacterium Chloroflexus aurantiacus.

The arrangement of core antenna complexes (B808-866-RC) in the cytoplasmic membrane of filamentous phototrophic bacterium Chloroflexus aurantiacus was studied by electron microscopy in cultures from different light conditions. A typical nearest-neighbor center-to-center distance of ~18 nm was found, implying less protein crowding compared to membranes of purple bacteria. A mean RC:chlorosome ratio of 11 was estimated for the occupancy of the membrane directly underneath each chlorosome, based on analysis of chlorosome dimensions and core complex distribution. Also presented are results of single-particle analysis of core complexes embedded in the native membrane.

Monitoring thylakoid ultrastructural changes in vivo using small-angle neutron scattering.

The light reactions of oxygenic photosynthesis take place in the thylakoid membranes, flattened vesicles, which contain the two photosystems and also embed the cytochrome b6f complex and the ATP synthase. In general, the thylakoid membranes are assembled into multilamellar membrane systems, which warrant an optimal light capturing efficiency. In nature, they show astounding variations, primarily due to large variations in their protein composition, which is controlled by multilevel regulatory mechanisms during long-term acclimation and short-term adaptation processes and also influenced by biotic or abiotic stresses - indicating a substantial degree of flexibility in the membrane ultrastructure. The better understanding of the dynamic features of this membrane system requires the use of non-invasive techniques, such as small angle neutron scattering (SANS), which is capable of providing accurate, statistically and spatially averaged information on the repeat distances of periodically organized thylakoid membranes under physiologically relevant conditions with time resolutions of seconds and minutes. In this review, after a short section on the basic properties of neutrons, we outline the fundamental principles of SANS measurements, its strengths and weaknesses in comparison to complementary structure investigation techniques. Then we overview recent results on isolated plant thylakoid membranes, and on living cyanobacterial and algal cells as well as on whole leaves. Special attention is paid to light-induced reversible ultrastructural changes in vivo, which, in cyanobacterial and diatom cells, were uncovered with the aid of SANS measurements; we also discuss the role of membrane reorganizations in light adaptation and photoprotection mechanisms.

Investigation of photosynthetic membrane structure using atomic force microscopy.

Photosynthetic processes, including light capture, electron transfer, and energy conversion, are not only ensured by the activities of individual photosynthetic complexes but also substantially determined and regulated by the composition and assembly of the overall photosynthetic apparatus at the supramolecular level. In recent years, atomic force microscopy (AFM) has matured as a unique and powerful tool for directly assessing the supramolecular assembly of integral membrane protein complexes in their native membrane environment at submolecular resolution. This review highlights the major contributions and advances of AFM studies to our understanding of the structure of the bacterial photosynthetic machinery and its regulatory arrangement during chromatic adaptation. AFM topographs of other biological membrane systems and potential future applications of AFM are also discussed.

Native architecture of the photosynthetic membrane from Rhodobacter veldkampii.

The photosynthetic membrane in purple bacteria contains several pigment-protein complexes that assure light capture and establishment of the chemiosmotic gradient. The bioenergetic tasks of the photosynthetic membrane require the strong interaction between these various complexes. In the present work, we acquired the first images of the native outer membrane architecture and the supramolecular organization of the photosynthetic apparatus in vesicular chromatophores of Rhodobacter (Rb.) veldkampii. Mixed with LH2 (light-harvesting complex 2) rings, the PufX-containing LH1-RC (light-harvesting complex 1--reaction center) core complexes appear as C-shaped monomers, with random orientations in the photosynthetic membrane. Within the LH1 fence surrounding the RC, a remarkable gap that is probably occupied (or partially occupied) by PufX is visualized. Sequence alignment revealed that one specific region in PufX may be essential for PufX-induced core dimerization. In this region of ten amino acids in length all Rhodobacter species had five conserved amino acids, with the exception of Rb. veldkampii. Our findings provide direct evidence that the presence of PufX in Rb. veldkampii does not directly govern the dimerization of LH1-RC core complexes in the native membrane. It is indicated, furthermore, that the high membrane curvature of Rb. veldkampii chromatophores (Rb. veldkampii features equally small vesicular chromatophores alike Rb. sphaeroides) is not due to membrane bending induced by dimeric RC-LH1-PufX cores, as it has been proposed in Rb. sphaeroides.

Quantum oscillatory exciton migration in photosynthetic reaction centers.

The harvesting of solar energy and its conversion to chemical energy is essential for all forms of life. The primary photon absorption, transport, and charge separation events, which trigger a chain of chemical reactions, take place in membrane-bound photosynthetic complexes. Whether quantum effects, stemming from entanglement of chromophores, persist in the energy transport at room temperature, despite the rapid decoherence effects caused by environment fluctuations, is under current active debate. If confirmed, these may explain the high efficiency of light harvesting and open up numerous applications to quantum computing and information processing. We present simulations of the photosynthetic reaction center of photosystem II that clearly establish oscillatory energy transport at room temperature originating from interference of quantum pathways. These signatures of quantum transport may be observed by two dimensional coherent optical spectroscopy.

Long-lived quantum coherence in photosynthetic complexes at physiological temperature.

Photosynthetic antenna complexes capture and concentrate solar radiation by transferring the excitation to the reaction center that stores energy from the photon in chemical bonds. This process occurs with near-perfect quantum efficiency. Recent experiments at cryogenic temperatures have revealed that coherent energy transfer--a wave-like transfer mechanism--occurs in many photosynthetic pigment-protein complexes. Using the Fenna-Matthews-Olson antenna complex (FMO) as a model system, theoretical studies incorporating both incoherent and coherent transfer as well as thermal dephasing predict that environmentally assisted quantum transfer efficiency peaks near physiological temperature; these studies also show that this mechanism simultaneously improves the robustness of the energy transfer process. This theory requires long-lived quantum coherence at room temperature, which never has been observed in FMO. Here we present evidence that quantum coherence survives in FMO at physiological temperature for at least 300 fs, long enough to impact biological energy transport. These data prove that the wave-like energy transfer process discovered at 77 K is directly relevant to biological function. Microscopically, we attribute this long coherence lifetime to correlated motions within the protein matrix encapsulating the chromophores, and we find that the degree of protection afforded by the protein appears constant between 77 K and 277 K. The protein shapes the energy landscape and mediates an efficient energy transfer despite thermal fluctuations.

Photosynthetic system in Blastochloris viridis revisited.

The bacterium Blastochloris viridis carries one of the simplest photosynthetic systems, which includes a single light-harvesting complex that surrounds the reaction center, membrane soluble quinones, and a soluble periplasmic protein cytochrome c(2) that shuttle between the reaction center and the bc(1) complex and act as electron carriers, as well as the ATP synthase. The close arrangement of the photosynthetic membranes in Bl. viridis, along with the extremely tight arrangement of the photosystems within these membranes, raises a fundamental question about the diffusion of the electron carriers. To address this issue, we analyzed the structure and response of the Bl. viridis photosynthetic system to various light conditions, by using a combination of electron microscopy, whole-cell cryotomography, and spectroscopic methods. We demonstrate that in response to high light intensities, the ratio of both cytochrome c(2) and bc(1) complexes to the reaction centers is increased. The shorter membrane stacks, along with the notion that the bc(1) complex is located at the highly curved edges of these stacks, result in a smaller average distance between the reaction centers and the bc(1) complexes, leading to shorter pathways of cytochrome c(2) between the two complexes. Under anaerobic conditions, the slow diffusion rate is further mitigated by keeping most of the quinone pool reduced, resulting in a concentration gradient of quinols that allows for a constant supply of theses electron carriers to the bc(1) complex.

Structural analysis of photosynthetic membranes by cryo-electron tomography of intact Rhodopseudomonas viridis cells.

During the photosynthetic process, highly organized membranal assemblies convert light into biochemical energy with high efficiency. We have used whole-mount cryo-electron tomography to study the intracellular architecture of the photosynthetic membranes of the anaerobic purple photosynthetic bacterium Rhodopseudomonas viridis, as well as the organization of the photosynthetic units within the membranes. Three-dimensional reconstruction demonstrates a continuity of the plasma membrane with the photosynthetic membranes that form tunnel-like structures with an average diameter of 31 nm+/-8 nm at the connection sites. The spacing between the photosynthetic membranes at their cytoplasmic faces was found to be 11 nm, thus enforcing a highly close packaging of the photosynthetic membranes. Analysis of successive tomographic slices allowed for derivation of the spacing between adjacent photosynthetic core complexes from a single-layered photosynthetic membrane, in situ. This analysis suggests that most, if not all, photosynthetic membranes in R. viridis are characterized by a similar two-dimensional hexagonal lattice organization.

From high-resolution AFM topographs to atomic models of supramolecular assemblies.

Atomic force microscopy (AFM) has developed into a powerful tool in membrane biology. AFM features an outstanding signal-to-noise ratio that allows substructures on individual macromolecules to be visualized. Most recently, AFM topographs have shown the supramolecular assembly of the bacterial photosynthetic complexes in native membranes. Here, we have determined the translational and rotational degrees of freedom of the complexes in AFM images of multi-protein assemblies, in order to build realistic atomic models of supramolecular assemblies by docking high-resolution structures into the topographs. Membrane protein assemblies of megadalton size comprising several hundreds of polypeptide chains and pigments were built with Angstrom precision.

EMatch: discovery of high resolution structural homologues of protein domains in intermediate resolution cryo-EM maps.

Cryo-EM has become an increasingly powerful technique for elucidating the structure, dynamics, and function of large flexible macromolecule assemblies that cannot be determined at atomic resolution. However, due to the relatively low resolution of cryo-EM data, a major challenge is to identify components of complexes appearing in cryo-EM maps. Here, we describe EMatch, a novel integrated approach for recognizing structural homologues of protein domains present in a 6-10 A resolution cryo-EM map and constructing a quasi-atomic structural model of their assembly. The method is highly efficient and has been successfully validated on various simulated data. The strength of the method is demonstrated by a domain assembly of an experimental cryo-EM map of native GroEL at 6 A resolution.

Evidences for interaction of PsbS with photosynthetic complexes in maize thylakoids.

The PsbS subunit of Photosystem II (PSII) has received much attention in the past few years, given its crucial role in photoprotection of higher plants. The exact location of this small subunit in thylakoids is also debated. In this work possible interaction partners of PsbS have been identified by immunoaffinity and immunoprecipitation, performed with mildly solubilized whole thylakoid membrane. The interacting proteins, as identified by mass spectrometry analysis of the immunoaffinity eluate, include CP29, some LHCII components, but also components of Photosystem I, of the cytochrome b(6)f complex as well as of ATP synthase. These proteins can be co-immunoprecipitated by using highly specific anti-PsbS antibodies and, vice-versa, PsbS is co-immunoprecipitated by antisera against components of the interacting complexes. We also find that PsbS co-migrates with bands containing PSII, ATP synthase and cytochrome b(6)f as well as with LHCII-containing bands on non-denaturing Deriphat PAGE. These results suggest multiple location of PsbS in the thylakoid membrane and point to an unexpected lateral mobility of this PSII subunit. As revealed by immunogold labelling with antibody against PsbS, the protein is associated either with granal membranes or prevalently with stroma lamellae in low or high-intensity light-treated intact leaves, respectively. This finding is consistent with the capability of PsbS to interact with complexes located in stroma lamellae, even though the exact physiological condition(s) under which these interactions may take place remain to be clarified.

Scanning electrochemical microscopy of the photosynthetic reaction center of Rhodobacter sphaeroides in different environmental systems.

The present work uses a scanning electrochemical microscopy technique to study systems containing the membrane-bound reaction center protein (RC) from the purple photosynthetic bacteria Rhodobacter spheroides to chromatophores (spherical reorganization of cell membrane following its mechanical rupture) and liposomes (reconstituted membrane systems at lower degree of complexity). Scanning electrochemical microscopy is a useful tool to investigate redox processes involving a RC, because the effective heterogeneous rate constants for the redox reaction with different mediators can be measured. The technique is also able to provide information on the role of the outer cell membrane permeation on the kinetics of the electron-transfer processes and to obtain more insight into the nature of the species involved.