Phosphoribulokinase abundance is not limiting the Calvin-Benson-Bassham cycle in Chlamydomonas reinhardtii.

Improving photosynthetic efficiency in plants and microalgae is of utmost importance to support the growing world population and to enable the bioproduction of energy and chemicals. Limitations in photosynthetic light conversion efficiency can be directly attributed to kinetic bottlenecks within the Calvin-Benson-Bassham cycle (CBBC) responsible for carbon fixation. A better understanding of these bottlenecks in vivo is crucial to overcome these limiting factors through bio-engineering. The present study is focused on the analysis of phosphoribulokinase (PRK) in the unicellular green alga Chlamydomonas reinhardtii. We have characterized a PRK knock-out mutant strain and showed that in the absence of PRK, Chlamydomonas cannot grow photoautotrophically while functional complementation with a synthetic construct allowed restoration of photoautotrophy. Nevertheless, using standard genetic elements, the expression of PRK was limited to 40% of the reference level in complemented strains and could not restore normal growth in photoautotrophic conditions suggesting that the CBBC is limited. We were subsequently able to overcome this initial limitation by improving the design of the transcriptional unit expressing PRK using diverse combinations of DNA parts including PRK endogenous promoter and introns. This enabled us to obtain strains with PRK levels comparable to the reference strain and even overexpressing strains. A collection of strains with PRK levels between 16% and 250% of WT PRK levels was generated and characterized. Immunoblot and growth assays revealed that a PRK content of ≈86% is sufficient to fully restore photoautotrophic growth. This result suggests that PRK is present in moderate excess in Chlamydomonas. Consistently, the overexpression of PRK did not increase photosynthetic growth indicating that that the endogenous level of PRK in Chlamydomonas is not limiting the Calvin-Benson-Bassham cycle under optimal conditions.

Blasticidin S Deaminase: A New Efficient Selectable Marker for Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii is a model unicellular organism for basic or biotechnological research, such as the production of high-value molecules or biofuels thanks to its photosynthetic ability. To enable rapid construction and optimization of multiple designs and strains, our team and collaborators have developed a versatile Chlamydomonas Modular Cloning toolkit comprising 119 biobricks. Having the ability to use a wide range of selectable markers is an important benefit for forward and reverse genetics in Chlamydomonas. We report here the development of a new selectable marker based on the resistance to the antibiotic blasticidin S, using the Bacillus cereus blasticidin S deaminase (BSR) gene. The optimal concentration of blasticidin S for effective selection was determined in both liquid and solid media and tested for multiple laboratory strains. In addition, we have shown that our new selectable marker does not interfere with other common antibiotic resistances: zeocin, hygromycin, kanamycin, paromomycin, and spectinomycin. The blasticidin resistance biobrick has been added to the Chlamydomonas Modular Cloning toolkit and is now available to the entire scientific community.

Nanovesicles released by Dictyostelium cells: a potential carrier for drug delivery.

Nanovesicles released by Dictyostelium discoideum cells grown in the presence of the DNA-specific dye Hoechst 33342 have been previously shown to mediate the transfer of the dye into the nuclei of Hoechst-resistant cells. The present investigation extends this work by conducting experiments in the presence of hypericin, a fluorescent therapeutic photosensitizer assayed for antitumoral photodynamic therapy. Nanovesicles released by Dictyostelium cells exhibit an averaged diameter between 50 and 150 nm, as measured by transmission cryoelectron microscopy. A proteomic analysis reveals a predominance of actin and actin-related proteins. The detection of a lysosomal membrane protein (LIMP II) indicates that these vesicles are likely generated in the late endosomal compartment. The use of the hypericin-containing nanovesicles as nanodevices for in vitro drug delivery was investigated by fluorescence microscopy. The observed signal was almost exclusively located in the perinuclear area of two human cell lines, skin fibroblasts (HS68) and cervix carcinoma (HeLa) cells. Studies by confocal microscopy with specific markers of cell organelles, provided evidence that hypericin was accumulated in the Golgi apparatus. All these data shed a new light on in vitro drug delivery by using cell-released vesicles as carriers.

The structure of phosphorylase kinase holoenzyme at 9.9 angstroms resolution and location of the catalytic subunit and the substrate glycogen phosphorylase.

Phosphorylase kinase (PhK) coordinates hormonal and neuronal signals to initiate the breakdown of glycogen. The enzyme catalyzes the phosphorylation of inactive glycogen phosphorylase b (GPb), resulting in the formation of active glycogen phosphorylase a. We present a 9.9 angstroms resolution structure of PhK heterotetramer (alphabetagammadelta)4 determined by cryo-electron microscopy single-particle reconstruction. The enzyme has a butterfly-like shape comprising two lobes with 222 symmetry. This three-dimensional structure has allowed us to dock the catalytic gamma subunit to the PhK holoenzyme at a location that is toward the ends of the lobes. We have also determined the structure of PhK decorated with GPb at 18 angstroms resolution, which shows the location of the substrate near the kinase subunit. The PhK preparation contained a number of smaller particles whose structure at 9.8 angstroms resolution was consistent with a proteolysed activated form of PhK that had lost the alpha subunits and possibly the gamma subunits.

Calcineurin B-like domains in the large regulatory alpha/beta subunits of phosphorylase kinase.

Phosphorylase kinase (PhK) is a large hexadecameric complex that catalyzes the phosphorylation and activation of glycogen phosphorylase (GP). It consists in four copies each of a catalytic subunit (gamma) and three regulatory subunits (alpha beta delta). Delta corresponds to endogenous calmodulin, whereas little is known on the molecular architecture of the large alpha and beta subunits, which probably arose from gene duplication. Here, using sensitive methods of sequence analysis, we show that the C-terminal domain (named domain D) of these alpha and beta subunits can be significantly related to calcineurin B-like (CBL) proteins. CBL are members of the EF-hand family that are involved in the regulation of plant-specific kinases of the CIPK/PKS family, and relieve autoinhibition of their target kinases by binding to their regulatory region. The relationship highlighted here suggests that PhK alpha and/or beta domain D may be involved in a similar regulation mechanism, a hypothesis which is supported by the experimental observation of a direct interaction between domain D of PhKalpha and the regulatory region of the Gamma subunit. This finding, together the identification of significant similarities of domain D with the preceding domain C, may help to understand the molecular mechanism by which PhK alpha and/or beta domain D might regulate PhK activity.

The subnanometer resolution structure of the glutamate synthase 1.2-MDa hexamer by cryoelectron microscopy and its oligomerization behavior in solution: functional implications.

The three-dimensional structure of the hexameric (alphabeta)(6) 1.2-MDa complex formed by glutamate synthase has been determined at subnanometric resolution by combining cryoelectron microscopy, small angle x-ray scattering, and molecular modeling, providing for the first time a molecular model of this complex iron-sulfur flavoprotein. In the hexameric species, interprotomeric alpha-alpha and alpha-beta contacts are mediated by the C-terminal domain of the alpha subunit, which is based on a beta helical fold so far unique to glutamate synthases. The alphabeta protomer extracted from the hexameric model is fully consistent with it being the minimal catalytically active form of the enzyme. The structure clarifies the electron transfer pathway from the FAD cofactor on the beta subunit, to the FMN on the alpha subunit, through the low potential [4Fe-4S](1+/2+) centers on the beta subunit and the [3Fe-4S](0/1+) cluster on the alpha subunit. The (alphabeta)(6) hexamer exhibits a concentration-dependent equilibrium with alphabeta monomers and (alphabeta)(2) dimers, in solution, the hexamer being destabilized by high ionic strength and, to a lower extent, by the reaction product NADP(+). Hexamerization seems to decrease the catalytic efficiency of the alphabeta protomer only 3-fold by increasing the K(m) values measured for l-Gln and 2-OG. However, it cannot be ruled out that the (alphabeta)(6) hexamer acts as a scaffold for the assembly of multienzymatic complexes of nitrogen metabolism or that it provides a means to regulate the activity of the enzyme through an as yet unknown ligand.

Structure of the acidianus filamentous virus 3 and comparative genomics of related archaeal lipothrixviruses.

Four novel filamentous viruses with double-stranded DNA genomes, namely, Acidianus filamentous virus 3 (AFV3), AFV6, AFV7, and AFV8, have been characterized from the hyperthermophilic archaeal genus Acidianus, and they are assigned to the Betalipothrixvirus genus of the family Lipothrixviridae. The structures of the approximately 2-mum-long virions are similar, and one of them, AFV3, was studied in detail. It consists of a cylindrical envelope containing globular subunits arranged in a helical formation that is unique for any known double-stranded DNA virus. The envelope is 3.1 nm thick and encases an inner core with two parallel rows of protein subunits arranged like a zipper. Each end of the virion is tapered and carries three short filaments. Two major structural proteins were identified as being common to all betalipothrixviruses. The viral genomes were sequenced and analyzed, and they reveal a high level of conservation in both gene content and gene order over large regions, with this similarity extending partly to the earlier described betalipothrixvirus Sulfolobus islandicus filamentous virus. A few predicted gene products of each virus, in addition to the structural proteins, could be assigned specific functions, including a putative helicase involved in Holliday junction branch migration, a nuclease, a protein phosphatase, transcriptional regulators, and glycosyltransferases. The AFV7 genome appears to have undergone intergenomic recombination with a large section of an AFV2-like viral genome, apparently resulting in phenotypic changes, as revealed by the presence of AFV2-like termini in the AFV7 virions. Shared features of the genomes include (i) large inverted terminal repeats exhibiting conserved, regularly spaced direct repeats; (ii) a highly conserved operon encoding the two major structural proteins; (iii) multiple overlapping open reading frames, which may be indicative of gene recoding; (iv) putative 12-bp genetic elements; and (v) partial gene sequences corresponding closely to spacer sequences of chromosomal repeat clusters.

High-resolution single-particle 3D analysis on GroEL prepared by cryo-negative staining.

Cryo-negative staining was developed as a complementary technique to conventional cryo-electron microscopy on supramolecular complexes. It allows imaging biological samples in a comparable state of structural preservation to conventional cryo-EM but the staining produces better contrast in accessible areas and allows data recording at lower defocus values. Cryo-negative staining vitrifies biological particles in the presence of a concentrated ammonium molybdate solution at neutral pH. It was successfully used to study the structure and dynamics of several macromolecules, such as human transcription factors and RNA polymerases. Imaging macromolecular complexes with cryo-negative staining has been established previously to better than 2 nm detail. However, it has not been verified so far whether cryo-negative staining also visualizes secondary structure elements. Using the well known E. coli GroEL chaperonin, we could show that the structure is well preserved to approximately 10 A resolution. Secondary structure details are at least partially resolved in the electron density map.

SPIDER image processing for single-particle reconstruction of biological macromolecules from electron micrographs.

This protocol describes the reconstruction of biological molecules from the electron micrographs of single particles. Computation here is performed using the image-processing software SPIDER and can be managed using a graphical user interface, termed the SPIDER Reconstruction Engine. Two approaches are described to obtain an initial reconstruction: random-conical tilt and common lines. Once an existing model is available, reference-based alignment can be used, a procedure that can be iterated. Also described is supervised classification, a method to look for homogeneous subsets when multiple known conformations of the molecule may coexist.

Structural basis for the PufX-mediated dimerization of bacterial photosynthetic core complexes.

In Rhodobacter (Rba.) sphaeroides, the subunit PufX is involved in the dimeric organization of the core complex. Here, we report the 3D reconstruction at 12 A by cryoelectron microscopy of the core complex of Rba. veldkampii, a complex of approximately 300 kDa without symmetry. The core complex is monomeric and constituted by a light-harvesting complex 1 (LH1) ring surrounding a uniquely oriented reaction center (RC). The LH1 consists of 15 resolved alpha/beta heterodimers and is interrupted. Within the opening, PufX polypeptide is assigned at a position facing the Q(B) site of the RC. This core complex is different from a dissociated dimer of the core complex of Rba. sphaeroides revealing that PufX in Rba. veldkampii is unable to dimerize. The absence in PufX of Rba. veldkampii of a G(31)XXXG(35) dimerization motif highlights the transmembrane interactions between PufX subunits involved in the dimerization of the core complexes of Rhodobacter species.

How ATP hydrolysis controls filament assembly from profilin-actin: implication for formin processivity.

Formins catalyze rapid filament growth from profilin-actin, by remaining processively bound to the elongating barbed end. The sequence of elementary reactions that describe filament assembly from profilin-actin at either free or formin-bound barbed ends is not fully understood. Specifically, the identity of the transitory complexes between profilin and actin terminal subunits is not known; and whether ATP hydrolysis is directly or indirectly coupled to profilin-actin assembly is not clear. We have analyzed the effect of profilin on actin assembly at free and FH1-FH2-bound barbed ends in the presence of ADP and non-hydrolyzable CrATP. Profilin blocked filament growth by capping the barbed ends in ADP and CrATP/ADP-Pi states, with a higher affinity when formin is bound. We confirm that, in contrast, profilin accelerates depolymerization of ADP-F-actin, more efficiently when FH1-FH2 is bound to barbed ends. To reconcile these data with effective barbed end assembly from profilin-MgATP-actin, the nature of nucleotide bound to both terminal and subterminal subunits must be considered. All data are accounted for quantitatively by a model in which a barbed end whose two terminal subunits consist of profilin-ATP-actin cannot grow until ATP has been hydrolyzed and Pi released from the penultimate subunit, thus promoting the release of profilin and allowing further elongation. Formin does not change the activity of profilin but simply uses it for its processive walk at barbed ends. Finally, if profilin release from actin is prevented by a chemical cross-link, formin processivity is abolished.

Analysis of tetramethylrhodamine-labeled actin polymerization and interaction with actin regulatory proteins.

The hydrolysis of ATP accompanying actin polymerization destabilizes the filament, controls actin assembly dynamics in motile processes, and allows the specific binding of regulatory proteins to ATP- or ADP-actin. However, the relationship between the structural changes linked to ATP hydrolysis and the functional properties of actin is not understood. Labeling of actin Cys374 by tetramethylrhodamine (TMR) has been reported to make actin non-polymerizable and enabled the crystal structures of ADP-actin and 5'-adenylyl beta,gamma-imidodiphosphate-actin to be solved. TMR-actin has also been used to solve the structure of actin in complex with the formin homology 2 domain of mammalian Dia1. To understand how the covalent modification of actin by TMR may affect the structural changes linked to ATP hydrolysis and to evaluate the functional relevance of crystal structures of TMR-actin in complex with actin-binding proteins, we have analyzed the assembly properties of TMR-actin and its interaction with regulatory proteins. We show that TMR-actin polymerized in very short filaments that were destabilized by ATP hydrolysis. The critical concentrations for assembly of TMR-actin in ATP and ADP were only an order of magnitude higher than those for unlabeled actin. The functional interactions of actin with capping proteins, formin, actin-depolymerizing factor/cofilin, and the VCA-Arp2/3 filament branching machinery were profoundly altered by TMR labeling. The data suggest that TMR labeling hinders the intramolecular movements of actin that allow its specific adaptative recognition by regulatory proteins and that determine its function in the ATP- or ADP-bound state.

Mutations in myosin heavy chain 11 cause a syndrome associating thoracic aortic aneurysm/aortic dissection and patent ductus arteriosus.

We have recently described two kindreds presenting thoracic aortic aneurysm and/or aortic dissection (TAAD) and patent ductus arteriosus (PDA) and mapped the disease locus to 16p12.2-p13.13 (ref. 3). We now demonstrate that the disease is caused by mutations in the MYH11 gene affecting the C-terminal coiled-coil region of the smooth muscle myosin heavy chain, a specific contractile protein of smooth muscle cells (SMC). All individuals bearing the heterozygous mutations, even if asymptomatic, showed marked aortic stiffness. Examination of pathological aortas showed large areas of medial degeneration with very low SMC content. Abnormal immunological recognition of SM-MHC and the colocalization of wild-type and mutant rod proteins in SMC, in conjunction with differences in their coimmunoprecipitation capacities, strongly suggest a dominant-negative effect. Human MYH11 gene mutations provide the first example of a direct change in a specific SMC protein leading to an inherited arterial disease.

Aggregation and gelation of micellar casein particles.

Micellar casein particles (submicelles) are formed by removing calcium phosphate from native casein. The submicelles aggregate and eventually form a gel with a rate that increases strongly with increasing temperature and casein concentration. At low casein concentrations the gel is very weak and collapses under its own weight so that a precipitate is formed. The structure of the aggregates is studied using light scattering and cryo-electron microscopy. It is found that the aggregates have a self-similar structure with fractal dimension 2. The viscoelastic properties of the gel are studied by frequency scans of the loss and storage moduli during the gelation process. The bonds between the submicelles probably involve calcium phosphate complexes.

Oligomeric assemblies of the Escherichia coli MalT transcriptional activator revealed by cryo-electron microscopy and image processing.

MalT, the dedicated transcriptional activator of the maltose regulon in Escherichia coli, is the prototype for a family of large (approximately 100 kDa) transcriptional activators. MalT self-association plays a key role in recognition of the target promoters, which contain several MalT sites that are cooperatively bound by the activator. The unliganded form of MalT is monomeric. The protein self-associates only in the presence of both ATP (or AMP-PNP, a non-hydrolysable analog of ATP) and maltotriose, the inducer. Here, we report cryo-electron microscopy analyses of MalT multimeric forms. We show that, in the presence of maltotriose and AMP-PNP, MalT associates into novel, polydisperse, curved homopolymers. The building block, corresponding to a MalT monomer, comprises an outer globular domain connected by a peduncle to an inner domain that mediates self-association. Image analyses highlight the significant conformational flexibility of these polymeric forms. In the presence of a DNA fragment containing a MalT-controlled promoter, malPp500, MalT forms homopolymers with a much smaller radius of curvature and a different conformation. We propose that MalT binding to the target promoters involves the assembly of a MalT homo-oligomer that is governed by the array of MalT sites present.

Structure of the filamentous phage pIV multimer by cryo-electron microscopy.

The homo-multimeric pIV protein constitutes a channel required for the assembly and export of filamentous phage across the outer membrane of Escherichia coli. We present a 22 A-resolution three-dimensional reconstruction of detergent-solubilized pIV by cryo-electron microscopy associated with image analysis. The structure reveals a barrel-like complex, 13.5 nm in diameter and 24 nm in length, with D14 point-group symmetry, consisting of a dimer of unit multimers. Side views of each unit multimer exhibit three cylindrical domains named the N-ring, the M-ring and the C-ring. Gold labeling of pIV engineered to contain a single cysteine residue near the N or C terminus unambiguously identified the N-terminal region as the N-ring, and the C-terminal region was inferred to make up the C-ring. A large pore, ranging in inner diameter from 6.0 nm to 8.8 nm, runs through the middle of the multimer, but a central domain, the pore gate, blocks it. Moreover, the pore diameter at the N-ring is smaller than the phage particle. We therefore propose that the pIV multimer undergoes a large conformational change during phage transport, with reorganization of the central domain to open the pore, and widening at the N-ring in order to accommodate the 6.5 nm diameter phage particle.

Three-dimensional architecture of the eukaryotic multisynthetase complex determined from negatively stained and cryoelectron micrographs.

This study provides the first description of the three-dimensional architecture of the multienzyme complex of aminoacyl-tRNA synthetases. Reconstructions were calculated from electron microscopic images of negatively stained and frozen hydrated samples using three independent angular assignment methods. In all cases, volumes show an asymmetric triangular arrangement of protein domains around a deep central cavity. The structures have openings or indentations on most sides. Maximum dimensions are ca. 19x16x10 nm. The central cavity is 4 nm in diameter and extends two-thirds of the length of the particle.

Three-dimensional structure of phosphorylase kinase at 22 A resolution and its complex with glycogen phosphorylase b.

Phosphorylase kinase (PhK) integrates hormonal and neuronal signals and is a key enzyme in the control of glycogen metabolism. PhK is one of the largest of the protein kinases and is composed of four types of subunit, with stoichiometry (alphabetagammadelta)(4) and a total MW of 1.3 x 10(6). PhK catalyzes the phosphorylation of inactive glycogen phosphorylase b (GPb), resulting in the formation of active glycogen phosphorylase a (GPa) and the stimulation of glycogenolysis. We have determined the three-dimensional structure of PhK at 22 A resolution by electron microscopy with the random conical tilt method. We have also determined the structure of PhK decorated with GPb at 28 A resolution. GPb is bound toward the ends of each of the lobes with an apparent stoichiometry of four GPb dimers per (alphabetagammadelta)(4) PhK. The PhK/GPb model provides an explanation for the formation of hybrid GPab intermediates in the PhK-catalyzed phosphorylation of GPb.

Striking conformational change suspected within the phosphoribulokinase dimer induced by interaction with GAPDH.

A multitechnique approach was used to study the [glyceraldehyde-3-phosphate dehydrogenase](2 x 4)-[phosphoribulokinase](2 x 2) multienzymatic complex of the alga Chlamydomonas reinhardtii. On the one hand, each component of the complex was compared with known atomic structures of related enzymes or of similar enzymes originating from different organisms. On the other hand, the overall low resolution architecture of the whole complex was studied using cryoelectron microscopy and image processing techniques. The dimers of phosphoribulokinase are suspected to undergo a dramatic change in activity during a cycle of binding and detaching from tetramers of glyceraldehyde-3-phosphate dehydrogenase. This is likely supported by strong structural differences between the modeled phosphoribulokinase dimers and the counterpart in the three-dimensional reconstruction volume of the whole complex obtained from cryoelectron microscope images.