Protein-based nanomaterials as protein heterogeneous nucleants and structural studies on enzymes from two photosynthetic organisms

Proteins, the most essential biological macromolecules, are involved in nearly every aspect of life. The elucidation of their three-dimensional structures through X-ray analysis has significantly contributed to our understanding of fundamental mechanisms in life processes. However, the obstacle of obtaining high-resolution protein crystals remains significant. Thus, searching for materials that can effectively induce nucleation of crystals is a promising and active field. This thesis work characterizes and prepares albumin nanoparticles as heterogeneous nucleants for protein crystallization. These stable Bovine Serum Albumin nanoparticles were synthesized via the desolvation method, purified efficiently, and characterized in terms of dimension, morphology, and secondary structure. The ability of BSA-NPs to induce macromolecule nucleation was tested on three model proteins, exhibiting significant results, with larger NPs inducing more nucleation. The second part of this work focuses on the structural study, mainly through X-ray crystallography, of five chloroplast and cytosolic enzymes involved in the fundamental cellular processes of two photosynthetic organisms, Chlamydomonas reinhardtii and Arabidopsis thaliana. The structures of three enzymes involved in the Calvin-Benson-Bassham Cycle, phosphoribulokinase, troseposphatisomerase, and ribulosiophosphate epimerase from Chlamydomonas reinhardtii, were solved to investigate their catalytic and regulatory mechanisms. Additionally, the structure of nitrosylated-CrTPI made it possible to identify Cys14 as a target for nitrosylation, and the crystallographic structure of CrRPE was solved for the first time, providing insights into its catalytic and regulatory properties. Finally, the structure of S-nitrosoglutathione reductase, AtGSNOR, was compared with that of AtADH1, revealing differences in their catalytic sites. Overall, seven crystallographic structures, including partially oxidized CrPRK, CrPRK/ATP, CrPRK/ADP/Ru5P, CrTPI-nitrosylated, apo-CrRPE, apo-AtGSNOR, and AtADH1-NADH, were solved and are yet to be deposited in the PDB.

Rapid evolution of sex-related genes in Chlamydomonas

Biological speciation ultimately results in prezygotic isolation—the inability of incipient species to mate with one another–but little is understood about the selection pressures and genetic changes that generate this outcome. The genus Chlamydomonas comprises numerous species of unicellular green algae, including numerous geographic isolates of the species C. reinhardtii. This diverse collection has allowed us to analyze the evolution of two sex-related genes: the mid gene of C. reinhardtii, which determines whether a gamete is mating-type plus or minus, and the fus1 gene, which dictates a cell surface glycoprotein utilized by C. reinhardtii plus gametes to recognize minus gametes. Low stringency Southern analyses failed to detect any fus1 homologs in other Chlamydomonas species and detected only one mid homolog, documenting that both genes have diverged extensively during the evolution of the lineage. The one mid homolog was found in C. incerta, the species in culture that is most closely related to C. reinhardtii. Its mid gene carries numerous nonsynonymous and synonymous codon changes compared with the C. reinhardtii mid gene. In contrast, very high sequence conservation of both the mid and fus1 sequences is found in natural isolates of C. reinhardtii, indicating that the genes are not free to drift within a species but do diverge dramatically between species. Striking divergence of sex determination and mate recognition genes also has been encountered in a number of other eukaryotic phyla, suggesting that unique, and as yet unidentified, selection pressures act on these classes of genes during the speciation process.

Estudo da influência de parâmetros ambientais (Fe e N) na produtividade lipídica da microalga Chlamydomonas sp.

Mestrado em Engenharia Química - Ramo Tecnologias de Protecção Ambiental

Sustainability Evaluation of Biodiesel Produced from Microalgae Chlamydomonas sp Grown in Brewery Wastewater

This study performs a sustainability evaluation of biodiesel from microalga Chlamydomonas sp. grown in 20 % (v/v) of brewery’s wastewater, blended with pentose sugars (xylose, arabinose or ribose resulting from the hydrolysis of brewer’s spent grains (BSG). The life cycle steps considered for the study are: microalgae cultivation, biomass processing and lipids extraction at the brewery site, and its conversion to biodiesel at a dedicated external biofuel’s plant. Three sustainability indicators (LCEE, FER and GW) were considered and calculated using experimental data. Literature data was used, whenever necessary, to complement life cycle data, thus allowing a more accurate sustainability evaluation. A comparative analysis of the biodiesel life cycle steps was also conducted, with the main goal of identifying which steps need to be improved. Results show that biomass processing, especially cell harvesting, microalgae cultivation, and lipids extraction are the main process bottlenecks. It is also analysed the influence on the microalgae biodiesel sustainability of adding each pentose sugar to the cultivation media, concluding that it strongly influences the biomass and lipid productivity. In particular, the addition of xylose is preferable in terms of lipid productivity, but from a sustainability point of view, ribose is the best, though the difference from xylose is not significant. Nevertheless, culture without pentose addition presents the best sustainability results.

Influência do nível hidrológico sobre a dieta de Leporinus reinhardtii (Characiformes, Anostomidae) em um reservatório do semiárido brasileiro

A Região Neotropical possui um elevado número de espécies de peixes de água doce, cujas adaptações para explorar diferentes tipos de alimento é particularmente evidente em reservatórios, refletindo numa considerável plasticidade alimentar. Characiformes é a mais diversificada ordem de peixes da região, tendo Anostomidae como uma das famílias mais especiosas, com destaque para Leporinus, com cerca de 80 espécies válidas. Embora suas espécies sejam consideradas primordialmente onívoras, algumas apresentam dieta herbívora, e a plasticidade alimentar do gênero é uma característica comum em ambientes sujeitos a variação sazonal de nível da água. No presente trabalho, são analisadas a composição da dieta de Leporinus reinhardtii Lütken, 1875 no reservatório de Sobradinho e a influência da variação sazonal de seu nível sobre a mesma ao longo de três ciclos anuais (2006-2009). Dentre os 876 indivíduos analisados, 302 foram coletados no período de seca e 574 no de cheia. A dieta foi composta por 47 itens, agrupados em nove categorias: Moluscos, Microcrustáceos, Insetos, Insetos (partes), Chironomidae, Vegetais, Sementes, Sedimento e Outros. Chironomidae representou em média 60% do peso total das categorias alimentares, seguido de Sementes com 25% e as demais categorias com menos de 10%. No período de cheia, nos trechos lêntico e de transição, predominaram Sementes na dieta, tendo Chironomidae sido mais representativa no trecho lótico, e as demais categorias não ultrapassando 20% de participação nos três trechos neste período. No período de seca, houve diferença nos itens predominantes, com predominância de Insetos no trecho lêntico, principalmente Coleoptera, e de Vegetais no lótico, com maior participação de macrófitas aquáticas, algas filamentosas, clorofíceas e da diatomácea Oscillatoria. Já no trecho de transição, Microcrustáceos dominaram na dieta, diferindo dos demais trechos. A dieta de L. reinhardtii se caracterizou por baixos valores de frequência de ocorrência de todas as categorias alimentares (predominantemente inferiores a 50%) e elevada abundância relativa (>80%) de diferentes categorias. A amplitude de nicho foi restrita nos diferentes trechos nos dois períodos, sendo ligeiramente mais elevada na cheia, devido à maior diversidade de itens, em particular no trecho lêntico. Leporinus reinhardtii possui hábito onívoro com comportamento especialista no reservatório de Sobradinho, cujas categorias predominantes da dieta dependem do regime hidrológico do reservatório e das características ambientais de cada trecho, refletindo numa amplitude restrita de nicho trófico para a espécie.

Águas com predominância de Eutreptia lanowi steuer e Chlamydomonas reinhardi dangeard no plancton, na enseada de Inhauma, Baía de Guanabara

In brackish waters of a creek of Guanabara Bay, the author points by the first time the presence of Chlamydomonas reinhardi, Eutreptia lanowi, Oscillatoria putrida, O. limosa, O. chlorina that were unknown in our waters; such biologic indicators proved themselves pollutional conditions, so bad a stark-mesosaprobic regime. Other news are plankton analysis by the Standar methods, of two most expressive samples of water masses;also the mobility of the plankters are measured in micra by second.

Cytoplasmic Dynein Heavy Chain 1b Is Required for Flagellar Assembly in Chlamydomonas

A second cytoplasmic dynein heavy chain (cDhc) has recently been identified in several organisms, and its expression pattern is consistent with a possible role in axoneme assembly. We have used a genetic approach to ask whether cDhc1b is involved in flagellar assembly in Chlamydomonas. Using a modified PCR protocol, we recovered two cDhc sequences distinct from the axonemal Dhc sequences identified previously. cDhc1a is closely related to the major cytoplasmic Dhc, whereas cDhc1b is closely related to the minor cDhc isoform identified in sea urchins, Caenorhabditis elegans, and Tetrahymena. The Chlamydomonas cDhc1b transcript is a low-abundance mRNA whose expression is enhanced by deflagellation. To determine its role in flagellar assembly, we screened a collection of stumpy flagellar (stf) mutants generated by insertional mutagenesis and identified two strains in which portions of the cDhc1b gene have been deleted. The two mutants assemble short flagellar stumps (<1–2 μm) filled with aberrant microtubules, raft-like particles, and other amorphous material. The results indicate that cDhc1b is involved in the transport of components required for flagellar assembly in Chlamydomonas.

The UNI3 Gene Is Required for Assembly of Basal Bodies of Chlamydomonas and Encodes δ-Tubulin, a New Member of the Tubulin Superfamily

We have cloned the UNI3 gene in Chlamydomonas and find that it encodes a new member of the tubulin superfamily. Although Uni3p shares significant sequence identity with α-, β-, and γ-tubulins, there is a region of Uni3p that has no similarity to tubulins or other known proteins. Mutant uni3–1 cells assemble zero, one, or two flagella. Pedigree analysis suggests that flagellar number in uni3–1 cells is a function of the age of the cell. The uniflagellate uni3–1 cells show a positional phenotype; the basal body opposite the eyespot templates the single flagellum. A percentage of uni3–1 cells also fail to orient the cleavage furrow properly, and basal bodies have been implicated in the placement of cleavage furrows in Chlamydomonas. Finally when uni3–1 cells are observed by electron microscopy, doublet rather than triplet microtubules are observed at the proximal end of the basal bodies. We propose that the Uni3 tubulin is involved in both the function and cell cycle-dependent maturation of basal bodies/centrioles.

The Mr 140,000 Intermediate Chain of Chlamydomonas Flagellar Inner Arm Dynein Is a WD-Repeat Protein Implicated in Dynein Arm Anchoring

Previous structural and biochemical studies have revealed that the inner arm dynein I1 is targeted and anchored to a unique site located proximal to the first radial spoke in each 96-nm axoneme repeat on flagellar doublet microtubules. To determine whether intermediate chains mediate the positioning and docking of dynein complexes, we cloned and characterized the 140-kDa intermediate chain (IC140) of the I1 complex. Sequence and secondary structural analysis, with particular emphasis on β-sheet organization, predicted that IC140 contains seven WD repeats. Reexamination of other members of the dynein intermediate chain family of WD proteins indicated that these polypeptides also bear seven WD/β-sheet repeats arranged in the same pattern along each intermediate chain protein. A polyclonal antibody was raised against a 53-kDa fusion protein derived from the C-terminal third of IC140. The antibody is highly specific for IC140 and does not bind to other dynein intermediate chains or proteins in Chlamydomonas flagella. Immunofluorescent microscopy of Chlamydomonas cells confirmed that IC140 is distributed along the length of both flagellar axonemes. In vitro reconstitution experiments demonstrated that the 53-kDa C-terminal fusion protein binds specifically to axonemes lacking the I1 complex. Chemical cross-linking indicated that IC140 is closely associated with a second intermediate chain in the I1 complex. These data suggest that IC140 contains domains responsible for the assembly and docking of the I1 complex to the doublet microtubule cargo.

The Chlamydomonas IDA7 Locus Encodes a 140-kDa Dynein Intermediate Chain Required to Assemble the I1 Inner Arm Complex

To identify new loci that are involved in the assembly and targeting of dynein complexes, we have screened a collection of motility mutants that were generated by insertional mutagenesis. One such mutant, 5B10, lacks the inner arm isoform known as the I1 complex. This isoform is located proximal to the first radial spoke in each 96-nm axoneme repeat and is an important target for the regulation of flagellar motility. Complementation tests reveal that 5B10 represents a new I1 locus, IDA7. Biochemical analyses confirm that ida7 axonemes lack at least five I1 complex subunits. Southern blots probed with a clone containing the gene encoding the 140-kDa intermediate chain (IC) indicate that the ida7 mutation is the result of plasmid insertion into the IC140 gene. Transformation with a wild-type copy of the IC140 gene completely rescues the mutant defects. Surprisingly, transformation with a construct of the IC140 gene lacking the first four exons of the coding sequence also rescues the mutant phenotype. These studies indicate that IC140 is essential for assembly of the I1 complex, but unlike other dynein ICs, the N-terminal region is not critical for its activity.

The Chlamydomonas PF6 Locus Encodes a Large Alanine/Proline-Rich Polypeptide That Is Required for Assembly of a Central Pair Projection and Regulates Flagellar Motility

Efficient motility of the eukaryotic flagellum requires precise temporal and spatial control of its constituent dynein motors. The central pair and its associated structures have been implicated as important members of a signal transduction cascade that ultimately regulates dynein arm activity. To identify central pair components involved in this process, we characterized a Chlamydomonas motility mutant (pf6-2) obtained by insertional mutagenesis. pf6-2 flagella twitch ineffectively and lack the 1a projection on the C1 microtubule of the central pair. Transformation with constructs containing a full-length, wild-type copy of the PF6 gene rescues the functional, structural, and biochemical defects associated with the pf6 mutation. Sequence analysis indicates that the PF6 gene encodes a large polypeptide that contains numerous alanine-rich, proline-rich, and basic domains and has limited homology to an expressed sequence tag derived from a human testis cDNA library. Biochemical analysis of an epitope-tagged PF6 construct demonstrates that the PF6 polypeptide is an axonemal component that cosediments at 12.6S with several other polypeptides. The PF6 protein appears to be an essential component required for assembly of some of these polypeptides into the C1-1a projection.

Molecular cloning of a protein kinase whose phosphorylation is regulated by genetic adhesion during Chlamydomonas fertilization.

Fertilization in Chlamydomonas is initiated by adhesive interactions between gametes of opposite mating types through flagellar glycoproteins called agglutinins. Interactions between these cell adhesion molecules signal for the activation of adenylyl cyclase through an interplay of protein kinases and ultimately result in formation of a diploid zygote. One of the early events during adhesion-induced signal transduction is the rapid inactivation of a flagellar protein kinase that phosphorylates a 48-kDa protein in the flagella. We report the biochemical and molecular characterization of the 48-kDa protein. Experiments using a bacterially expressed fusion protein show that the 48-kDa protein is capable of autophosphorylation on serine and tyrosine and phosphorylation of bovine beta-casein on serine, confirming that the 48-kDa protein itself has protein kinase activity. This protein kinase exhibits limited homology to members of the eukaryotic protein kinase superfamily and may be an important element in a signaling pathway in fertilization.

Structural basis for control of secondary vessels in the long-finned eel, Anguilla reinhardtii

Histological sections of primary segmental arteries and associated interarterial anastomoses and secondary vessels from the long-finned eel Anguilla reinhardtii were examined by light and transmission electron microscopy. Interarterial anastomoses were found to originate from the primary vasculature as depressions through the tunica intima and media, from where they ran perpendicularly to the adventitial layer, before coiling extensively. From here the anastomoses travelled a relatively linear path in the outer margin of the adventitia to anastomose with a secondary vessel running in parallel with the primary counterpart. In contrast to findings from other species, secondary vessels had a structure quite similar to that of primary vessels; they were lined by endothelial cells on a continuous basement membrane, with a single layer of smooth muscle cells surrounding the vessel. Smooth muscle cells were also found in the vicinity of interarterial anastomoses in the adventitia, but these appeared more longitudinally orientated. The presence of smooth muscle cells on all aspects of the secondary circulation suggests that this vascular system is regulated in a similar manner as the primary vascular system. Because interarterial anastomoses are structurally integrated with the primary vessel from which they originate, it is anticipated that flow through secondary vessels to some extent is affected by the vascular tone of the primary vessel. Immunohistochemical studies showed that primary segmental arteries displayed moderate immunoreactivity to antibodies against 5-hydroxytryptamine and substance P, while interarterial anastomoses and secondary vessels showed dense immunoreactivity. No immunoreactivity was observed on primary or secondary arteries against neuropeptide Y or calcitonin gene-related peptide.

The secondary vascular system of Actinopterygii: interspecific variation in origins and investment

Vascular casts of 3 species of Chondrichthyes, 1 of Dipnoi, 1 of Chondrostei and 14 species of the Teleostei were examined by light and scanning electron microscopy in order to give a qualitative and quantitative analysis of interarterial anastomoses (iaas) that indicate the presence (or absence) of a secondary vascular system (SVS). Anastomoses were found to originate from a variety of different primary blood vessels, many of which have not been previously identified as giving rise to secondary vessels. Segmental arteries derived from the dorsal aorta and supplying body musculature were major sites of origin of the SVS, although there was considerable variation in where, in the hierarchy of arterial branching, the anastomoses occurred. The degree of investment in a SVS was species specific, with more active species having a higher degree of secondary vascularisation. This difference was quantified using an absolute count of iaas between Anguilla reinhardtii and Trachinotus baillonii. A range of general features of the SVS is also described. No evidence of iaas was found on the coeliac, mesenteric or renal circulation in any species. Evidence of iaas was lacking in the dipnoan and chondrichthyan species examined, suggesting that a SVS is restricted to Actinopterygii. The presence and distribution of a SVS does not appear to be exclusively linked to phylogenetic position, but rather to the physiological adaptation of the species.