Crystallization of the photosystem I reaction centre

The reaction centre of the photosynthetic membrane complex photosystem I (PSI) from the thermophilic cyanobacterium Phormidium laminosum was found to crystallize under a range of conditions. The crystallization method, which can occur in the presence of larger detergent molecules than those used previously for the crystallization of membrane proteins, is presented in this report. Several crystal forms have been observed, and some of these show birefringence and linear dichroism. Optical measurements on crystals thicker than ˜5 µm were severely restricted because of the very high chlorophyll density within the crystals, but linear dichroism measurements on thin single crystals were possible and the results are presented here. By comparing the data with earlier measurements on oriented PSI complexes, a working model for the orientation of the PSI complexes within the crystal could be proposed. The PSI reaction centre is one of the largest and most complex membrane protein units that have been crystallized to date.     

Organization and role of the long-wave chlorophylls in the photosystem I of the Cyanobacterium spirulina.

The data on the organization and function of the photosystem I pigment-protein complexes of the cyanobacterium Spirulina and the characteristics of pigment antenna of the photosystem I monomeric and trimeric core complexes are presented and discussed. We proved that the photosystem I complexes in the cyanobacterial membrane pre-exist mainly as trimers, though both types of complexes contribute to the photosynthetic electron transport. In contrast to monomers, the antenna of the photosystem I trimeric complexes of Spirulina contains the extreme long-wave chlorophyll form absorbing at 735 nm and emitting at 760 nm (77 K). The intensity of fluorescence at 760 nm depends strongly on the P700 redox state: it is maximum with the reduced P700 and strongly decreased with the oxidized P700 which is the most efficient quencher of fluorescence at 760 nm. The energy absorbed by the extreme long-wave chlorophyll form is active in the photooxidation of P700 in the trimeric complex. The data obtained indicate that the long-wave form of chlorophyll originates from interaction of the chlorophyll molecules localized on monomeric subunits forming the photosystem I trimer. Kinetic analysis of the P700 photooxidation and light-induced quenching of fluorescence at 760 nm (77 K) allows the suggestion that the excess energy absorbed by the antenna monomeric subunits within the trimer migrates via the extreme long-wave chlorophyll to the P700 cation radical and is quenched, which prevents the photodestruction of the pigment-protein complex.     

Thermal stability of trimeric light-harvesting chlorophyll a/b complex (LHCIIb) in liposomes of thylakoid lipids

The major light-harvesting chlorophyll a/b complex (LHCIIb) of photosystem (PS) II functions by harvesting light energy and by limiting and balancing the energy flow directed towards the PSI and PSII reaction centers. The complex is predominantly trimeric; however, the monomeric form may play a role in one or several of the regulatory functions of LHCIIb. In this work the dissociation temperature was measured of trimeric LHCIIb isolated from Pisum thylakoids and inserted into liposomes made of various combinations of thylakoid lipids at various protein densities. Dissociation was measured by monitoring a trimer-specific circular dichroism signal in the visible range. The LHCIIb density in the membrane significantly affected the trimer dissociation temperature ranging from 70 °C at an LHCIIb concentration comparable to or higher than the one in thylakoid grana, to 65 °C at the density estimated in stromal lamellae. Omitting one thylakoid lipid from the liposomes had virtually no effect on the thermal trimer stability in most cases except when digalactosyl diacylglycerol (DGDG) was omitted which caused a drop in the apparent dissociation temperature by 2 °C. In liposomes containing only one lipid species, DGDG and, even more so, monogalactosyl diacylglycerol (MGDG) increased the thermal stability of LHCIIb trimers whereas phosphatidyl diacylglycerol (PG) significantly decreased it. The lateral pressure exerted by the non-bilayer lipid MGDG did not significantly influence LHCII trimer stability.     

In vitro oligomerization of a membrane protein complex. liposome-based reconstitution of trimeric photosystem I from isolated monomers.

Many membrane proteins can be isolated in different oligomeric forms. Photosystem I (PSI), for example, exists in cyanobacteria either as a monomeric or as a trimeric complex. Neither the factors responsible for the specific trimerization process nor its biological role are known at present. In the filamentous cyanobacterium Spirulina platensis, trimers in contrast to monomers show chlorophyll fluorescence emission at 760 nm. To investigate the oligomerization process as well as the nature of the long wavelength chlorophylls, we describe here an in vitro reconstitution procedure to assemble trimeric PS I from isolated purified PS I monomers. Monomers (and trimers) were extracted from S. platensis with n-dodecyl beta-D-maltoside and further purified by perfusion chromatography steps. The isolated complexes had the same polypeptide composition as other cyanobacteria (PsaA-PsaF and PsaI-PsaM), as determined from high resolution gels and immunoblotting. They were incorporated into proteoliposomes, which had been prepared by the detergent absorption method, starting from a phosphatidylcholine:phosphatidic acid mixture solubilized by octylglucoside. After the addition of monomeric PS I (lipid:chlorophyll, 25:1), octylglucoside was gradually removed by the stepwise addition of Biobeads. The 77 K fluorescence emission spectrum of these proteoliposomes displays a long wavelength emission at 760 nm that is characteristic of PS I trimers, which indicates for the first time the successful in vitro reconstitution of PS I trimers. In addition, a high performance liquid chromatography analysis of complexes extracted from these proteoliposomes confirms the formation of structural trimers. We also could show with this system 1) that at least one of the stromal subunits PsaC, -D, and -E is necessary for trimer formation and 2) that the extreme long wavelength emitting chlorophyll is formed as a result of trimer formation.     

Thermal stability of trimeric light-harvesting chlorophyll a/b complex (LHCIIb) in liposomes of thylakoid lipids

The major light-harvesting chlorophyll a/b complex (LHCIIb) of photosystem (PS) II functions by harvesting light energy and by limiting and balancing the energy flow directed towards the PSI and PSII reaction centers. The complex is predominantly trimeric; however, the monomeric form may play a role in one or several of the regulatory functions of LHCIIb. In this work the dissociation temperature was measured of trimeric LHCIIb isolated from Pisum thylakoids and inserted into liposomes made of various combinations of thylakoid lipids at various protein densities. Dissociation was measured by monitoring a trimer-specific circular dichroism signal in the visible range. The LHCIIb density in the membrane significantly affected the trimer dissociation temperature ranging from 70 degrees C at an LHCIIb concentration comparable to or higher than the one in thylakoid grana, to 65 degrees C at the density estimated in stromal lamellae. Omitting one thylakoid lipid from the liposomes had virtually no effect on the thermal trimer stability in most cases except when digalactosyl diacylglycerol (DGDG) was omitted which caused a drop in the apparent dissociation temperature by 2 degrees C. In liposomes containing only one lipid species, DGDG and, even more so, monogalactosyl diacylglycerol (MGDG) increased the thermal stability of LHCIIb trimers whereas phosphatidyl diacylglycerol (PG) significantly decreased it. The lateral pressure exerted by the non-bilayer lipid MGDG did not significantly influence LHCII trimer stability.     

Engineered oligomerization state of OmpF protein through computational design decouples oligomer dissociation from unfolding

Biogenesis of β-barrel membrane proteins is a complex, multistep, and as yet incompletely characterized process. The bacterial porin family is perhaps the best-studied protein family among β-barrel membrane proteins that allows diffusion of small solutes across the bacterial outer membrane. In this study, we have identified residues that contribute significantly to the protein-protein interaction (PPI) interface between the chains of outer membrane protein F (OmpF), a trimeric porin, using an empirical energy function in conjunction with an evolutionary analysis. By replacing these residues through site-directed mutagenesis either with energetically favorable residues or substitutions that do not occur in natural bacterial outer membrane proteins, we succeeded in engineering OmpF mutants with dimeric and monomeric oligomerization states instead of a trimeric oligomerization state. Moreover, our results suggest that the oligomerization of OmpF proceeds through a series of interactions involving two distinct regions of the extensive PPI interface: two monomers interact to form a dimer through the PPI interface near G19. This dimer then interacts with another monomer through the PPI interface near G135 to form a trimer. We have found that perturbing the PPI interface near G19 results in the formation of the monomeric OmpF only. Thermal denaturation of the designed dimeric OmpF mutant suggests that oligomer dissociation can be separated from the process of protein unfolding. Furthermore, the conserved site near G57 and G59 is important for the PPI interface and might provide the essential scaffold for PPIs.     

Lipid Fingerprints and Cofactor Dynamics of Light-Harvesting Complex II in Different Membranes

Plant light-harvesting complex II (LHCII) is the key antenna complex for plant photosynthesis. We present coarse-grained molecular dynamics simulations of monomeric and trimeric LHCII in a realistic thylakoid membrane environment based on the Martini force field. The coarse-grained protein model has been optimized with respect to atomistic reference simulations. Our simulations provide detailed insights in the thylakoid lipid fingerprint of LHCII which compares well with experimental data from membrane protein purification. Comparing the monomer and trimeric LHCII reveals a stabilizing effect of trimerization on the chromophores as well as the protein. Moreover, the average chromophore distance shortens in the trimer leading to stronger excitonic couplings. When changing the native thylakoid environment to a model membrane the protein flexibility remains constant, whereas the chromophore flexibility is reduced. Overall, the presented LHCII model lays the foundation to investigate the μs dynamics of this key antenna protein of plants.     

The preparation and characterisation of native photoreceptor units from the thylakoids of Rhodopseudomonas viridis

The photosynthetic membranes of Rhodopseudomonas viridis consist of a regular array of structural units. Each unit is composed of a central core (thought to contain the reaction centre complex) surrounded by a subdivided ring of protein (of likely antennae function). These individual units can be dissociated from the membrances using a variety of detergent treatments. The absorption spectrum, used as a criterion of a native state, is retained. All of the seven major polypeptides, the four reaction centre polypeptides (cytochrome, H, M and L chain) as well as the three light-harvesting polypeptides (B1015-α, β and ξ) are shown to be present. Electron microscopy of the units shows a similar structure to the units within the membrane. surface-specific iodination of both membranes and units labels predominantly polypeptides H, B1015-α, and ξ. M and L are weakly labelled. In addition, B1015-β is labelled in the isolated units. This, with other evidence, supports an allocation of light-harvesting polypeptides to the outer ring. Further solubilisation of these units separates the reaction centre (as a native complex containing all four polypeptides) from the light-harvesting polypeptides. The light-harvesting polypeptides are obtained in a form containing all three polypeptides and bound pigment, however the peak at 1015 nm corresponding to native bacteriochlorophyll b is lost.     

Affinity Enhancement by Ligand Clustering Effect Inspired by Peptide Dendrimers?Shank PDZ Proteins Interactions

High-affinity binders are desirable tools to probe the function that specific protein−protein interactions play in cell. In the process of seeking a general strategy to design high-affinity binders, we found a clue from the βPIX (p21-activated kinase interacting exchange factor)−Shank PDZ interaction in synaptic assembly: three PDZ-binding sites are clustered by a parallel coiled-coil trimer but bind to Shank PDZ protein with 1:1 stoichiometry (1 trimer/1 PDZ). Inspired by this architecture, we proposed that peptide dendrimer, mimicking the ligand clustering in βPIX, will also show enhanced binding affinity, yet with 1:1 stoichiometry. This postulation has been proven here, as we synthesized a set of monomeric, dimeric and trimeric peptides and measured their binding affinity and stoichiometry with Shank PDZ domains by isothermal titration calorimetry, native mass spectrometry and surface plasmon resonance. This affinity enhancement, best explained by proximity effect, will be useful to guide the design of high-affinity blockers for protein−protein interactions.     

Evolutionary Conservation in Biogenesis of β-Barrel Proteins Allows Mitochondria to Assemble a Functional Bacterial Trimeric Autotransporter Protein

Yersinia adhesin A (YadA) belongs to a class of bacterial adhesins that form trimeric structures. Their mature form contains a passenger domain and a C-terminal β-domain that anchors the protein in the outer membrane (OM). Little is known about how precursors of such proteins cross the periplasm and assemble into the OM. In the present study we took advantage of the evolutionary conservation in the biogenesis of β-barrel proteins between bacteria and mitochondria. We previously observed that upon expression in yeast cells, bacterial β-barrel proteins including the transmembrane domain of YadA assemble into the mitochondrial OM. In the current study we found that when expressed in yeast cells both the monomeric and trimeric forms of full-length YadA were detected in mitochondria but only the trimeric species was fully integrated into the OM. The oligomeric form was exposed on the surface of the organelle in its native conformation and maintained its capacity to adhere to host cells. The co-expression of YadA with a mitochondria-targeted form of the bacterial periplasmic chaperone Skp, but not with SurA or SecB, resulted in enhanced levels of both forms of YadA. Taken together, these results indicate that the proper assembly of trimeric autotransporter can occur also in a system lacking the lipoproteins of the BAM machinery and is specifically enhanced by the chaperone Skp.     

A trimeric DNA polymerase complex increases the native replication processivity

DNA polymerases are essential enzymes in all domains of life for both DNA replication and repair. The primary DNA replication polymerase from Sulfolobus solfataricus (SsoDpo1) has been shown previously to provide the necessary polymerization speed and exonuclease activity to replicate the genome accurately. We find that this polymerase is able to physically associate with itself to form a trimer and that this complex is stabilized in the presence of DNA. Analytical gel filtration and electrophoretic mobility shift assays establish that initially a single DNA polymerase binds to DNA followed by the cooperative binding of two additional molecules of the polymerase at higher concentrations of the enzyme. Protein chemical crosslinking experiments show that these are specific polymerase–polymerase interactions and not just separate binding events along DNA. Isothermal titration calorimetry and fluorescence anisotropy experiments corroborate these findings and show a stoichiometry where three polymerases are bound to a single DNA substrate. The trimeric polymerase complex significantly increases both the DNA synthesis rate and the processivity of SsoDpo1. Taken together, these results suggest the presence of a trimeric DNA polymerase complex that is able to synthesize long DNA strands more efficiently than the monomeric form.     

Demonstration of a folded monomeric form of porin PhoE of Escherichia coli in vivo.

The porins in the outer membranes of gram-negative bacteria are trimeric proteins. A folded monomeric form of the Escherichia coli porin PhoE, with a higher electrophoretic mobility than that of the denatured protein, has recently been detected in in vitro folding studies. To investigate the possible biological significance of the folded monomer, we attempted to detect this form in vivo. After pulse-labeling, folded monomers could be detected by immunoprecipitation. Furthermore, folded monomers were detected in a preparation of mutant PhoE porins, in which the subunit interactions were weakened by a E-66-->R substitution. Together, these results show that the folded monomer is not an in vitro folding artifact but an integral part of the native trimer.     

Biochemical and biophysical characterization of OmpG: A monomeric porin

A recombinant form of the porin OmpG, OmpGm, lacking the signal sequence, has been expressed in Escherichia coli. After purification under denaturing conditions, the protein was refolded in the detergent Genapol X-080, where it gained a structure rich in beta sheet as evidenced by a CD spectrum similar to that of the native form. Electrophoretic analysis and limited proteolysis experiments suggested that refolded OmpGm exists in at least three forms. Nevertheless, the recombinant protein formed uniform channels in planar bilayers with a conductance of 0.81 nS (1 M NaCl, pH 7.5). Previous biochemical studies had suggested that OmpG is a monomeric porin, rather than the usual trimer. Bilayer recordings substantiated this proposal; voltage-induced closures occurred consistently in a single step, and channel block by Gd(3+) lacked the cooperativity seen with the trimeric porin OmpF. The availability of milligram amounts of a monomeric porin will be useful both for basic studies of porin function and for membrane protein engineering.     

The TolB protein interacts with the porins of Escherichia coli.

TolB is a periplasmic protein of the cell envelope Tol complex. It is partially membrane associated through an interaction with the outer membrane lipoprotein PAL (peptidoglycan-associated lipoprotein), which also belongs to the Tol system. The interaction of TolB with outer membrane porins of Escherichia coli was investigated with a purified TolB derivative harboring a six-histidine tag. TolB interacted with the trimeric porins OmpF, OmpC, PhoE, and LamB but not with their denatured monomeric forms or OmpA. These interactions took place both in the presence and in the absence of lipopolysaccharide. TolA, an inner membrane component of the Tol system, also interacts with the trimeric porins via its central periplasmic domain (R. Dérouiche, M. Gavioli, H. Bénédetti, A. Prilipov, C. Lazdunski, and R. Lloubès, EMBO J. 15:6408-6415, 1996). In the presence of the purified central domain of TolA (TolAIIHis), the TolB-porin complexes disappeared to form TolAIIHis-porin complexes. These results suggest that the interactions of TolA and TolB with porins might take place in vivo and might be concomitant events participating in porin assembly. They also suggest that the Tol system as a whole may be involved in porin assembly in the outer membrane.     

假根羽藻主要捕光叶绿素a/b-蛋白复合体的特性

应用阴离子交换和凝胶过滤层析技术,从假根羽藻(Bryopsis corticulans Setch.)类囊体膜中直接分离、纯化获得了主要叶绿素a/b-蛋白复合体(LHCⅡ).经蔗糖密度梯度超速离心获得了该色素蛋白复合体的单体和三聚体.反相液相色谱的色素分析结果显示,假根羽藻LHCⅡ的色素组成含有叶绿素a、叶绿素b、新黄质、紫黄质和管藻素等.其单体的电子跃迁能谱与三聚体的相似.园二色光谱分析显示,在LHCⅡ脱辅基蛋白质上分别存在着很强的叶绿素a偶极子之间和叶绿素b偶极子之间的分子内相互作用,然而这些偶极子之间的分子间的相互作用在三聚体中得到明显增强.在能量传递方面,LHCⅡ单体有着与三聚体相似的从叶绿素b到叶绿素a以及从管藻素到叶绿素a的高效传能能力.实验结果表明,假根羽藻中LHCⅡ单体具有像三聚体那样可以高效发挥吸能和传能生理功能的色素组成形式.因此,这些单体可能是假根羽藻类囊体膜上具有功能作用的LHCⅡ的结构形式.     

Structure of Double-Shelled Rice Dwarf Virus

Rice dwarf virus (RDV), a member of the Reoviridae family, is a double-stranded RNA virus. Infection of rice plants with RDV reduces crop production significantly and can pose a major economic threat to Southeast Asia. A 25-Å three-dimensional structure of the 700-Å-diameter RDV capsid has been determined by 400-kV electron cryomicroscopy and computer reconstruction. The structure revealed two distinctive icosahedral shells: a T=13l outer icosahedral shell composed of 260 trimeric clusters of P8 (46 kDa) and an inner T=1 icosahedral shell of 60 dimers of P3 (114 kDa). Sequence and structural comparisons were made between the RDV outer shell trimer and the two crystal conformations (REF and HEX) of the VP7 trimer of bluetongue virus, an animal analog of RDV. The low-resolution structural match of the RDV outer shell trimer to the HEX conformation of VP7 trimer has led to the proposal that P8 consists of an upper domain of β-sandwich motif and a lower domain of α helices. The less well fit REF conformation of VP7 to the RDV trimer may be due to the differences between VP7 and P8 in the sequence of the hinge region that connects the two domains. The additional mass density and the absence of a known signaling peptide on the surface of the RDV outer shell trimer may be responsible for the different interactions between plants and animal reoviruses.     

Quaternary structure of influenza virus hemagglutinin after acid treatment.

Hemagglutinin (HA), a trimeric spike glycoprotein of influenza virus, mediates fusion between the viral envelope and the membrane of an endosome during virus entry. Fusion is triggered by low pH, which induces an irreversible conformational change in the protein. Several studies have indicated that intersubunit contacts along the trimer interfaces may be broken during this alteration. To determine whether HA dissociates into individual subunits as a consequence of the conformational change, we used velocity gradient sedimentation in the presence of Triton X-100. We also determined the resistance of acid-treated HA to dissociation by sodium dodecyl sulfate, a property of the HA trimer. At pH 7.0, isolated HA sedimented as a 9S trimer and gave the characteristic trimer pattern after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After acidification the HA remained trimeric irrespective of whether it was exposed to acid in intact virus particles or in solubilized form. Only when very low concentrations of HA were acidified did a fraction dissociate to dimers and monomers. In contrast, the water-soluble ectodomain fragment of HA (BHA) readily dissociated under a variety of conditions. Negative-stain electron microscopy supported the notion that HA molecules in virus particles do not dissociate upon acidification and may form larger oligomeric structures in the plane of the viral membrane. Taken together, the results suggested that it is the trimeric HA, or higher-order structures thereof, that are active in the acid-induced fusion reaction. Further, the results emphasized the role of the transmembrane anchors of HA in preventing dissociation of the trimer.     

A new putative cellulose-synthesizing complex ofColeochaete scutata

Summary Cells of the charophycean alga,Coleochaete scutata active in cell wall formation were freeze fractured in the search for cellulose synthesizing complexes (TCs) since this alga is considered to be among the most advanced and a progenitor to land plant evolution. We have found a new TC which consists of two geometrically distinctive particle complexes complementary to one another in the plasma membrane and occasionally associated with microfibril impressions. In the E-fracture face is found a cluster of 8–50 closely packed particles, each with a diameter of 5–17 nm. Most of these particles are confined within an 80 nm circle. In the P-fracture face is found an 8-fold symmetrical arrangement of 10 nm particles circumferentially arranged around a 28 nm central particle. The TCs ofC. scutata are quite distinctive from the rosette/globule TCs of land plants. The 5.5×3.1 nm microfibril inC. scutata is also distinctive from the 3.5×3.5 nm microfibril typical of land plants. The phylogenetic implications of this unique TC in land plant evolution are discussed.