Eduardo A. Ceccarelli

Bio: Eduardo A. Ceccarelli is an academic researcher from National University of Rosario. The author has contributed to research in topics: Ferredoxin—NADP(+) reductase & Ferredoxin. The author has an hindex of 28, co-authored 82 publications receiving 4039 citations. Previous affiliations of Eduardo A. Ceccarelli include National Scientific and Technical Research Council & University of California, San Diego.

Recombinant protein expression in Escherichia coli: advances and challenges.

Abstract: Escherichia coli is the organism of choice for the production of recombinant proteins. Its use as a cell factory is well-established and it has become the most popular expression platform. For this reason, there are many molecular tools and protocols at hand for the high-level production of recombinant proteins, such as a vast catalog of expression plasmids, a great number of engineered strains and many cultivation strategies. We review the different approaches for the synthesis of recombinant proteins in E. coli and discuss recent progress in this ever-growing field.

Open questions in ferredoxin‐NADP+ reductase catalytic mechanism

Abstract: Ferredoxin (flavodoxin)-NADP(H) reductases (FNR) are ubiquitous flavoenzymes that deliver NADPH or low potential one-electron donors (ferredoxin, flavodoxin) to redox-based metabolisms in plastids, mitochondria and bacteria. The plant-type reductase is also the basic prototype for one of the major families of flavin-containing electron transferases that display common functional and structural properties. Many aspects of FNR biochemistry have been extensively characterized in recent years using a combination of site-directed mutagenesis, steady-state and transient kinetic experiments, spectroscopy and X-ray crystallography. Despite these considerable advances, various key features in the enzymology of these important reductases remain yet to be explained in molecular terms. This article reviews the current status of these open questions. Measurements of electron transfer rates and binding equilibria indicate that NADP(H) and ferredoxin interactions with FNR result in a reciprocal decrease of affinity, and that this induced-fit step is a mandatory requisite for catalytic turnover. However, the expected conformational movements are not apparent in the reported atomic structures of these flavoenzymes in the free state or in complex with their substrates. The overall reaction catalysed by FNR is freely reversible, but the pathways leading to NADP+ or ferredoxin reduction proceed through entirely different kinetic mechanisms. Also, the reductases isolated from various sources undergo inactivating denaturation on exposure to NADPH and other electron donors that reduce the FAD prosthetic group, a phenomenon that might have profound consequences for FNR function in vivo. The mechanisms underlying this reductive inhibition are so far unknown. Finally, we provide here a rationale to interpret FNR evolution in terms of catalytic efficiency. Using the formalism of the Albery–Knowles theory, we identified which parameter(s) have to be modified to make these reductases even more proficient under a variety of conditions, natural or artificial. Flavoenzymes with FNR activity catalyse a number of reactions with potential importance for biotechnological processes, so that modification of their catalytic competence is relevant on both scientific and technical grounds.

New tools for recombinant protein production in Escherichia coli: A 5-year update.

Abstract: The production of proteins in sufficient amounts is key for their study or use as biotherapeutic agents. Escherichia coli is the host of choice for recombinant protein production given its fast growth, easy manipulation, and cost-effectiveness. As such, its protein production capabilities are continuously being improved. Also, the associated tools (such as plasmids and cultivation conditions) are subject of ongoing research to optimize product yield. In this work, we review the latest advances in recombinant protein production in E. coli.

A productive NADP+ binding mode of ferredoxin-NADP+ reductase revealed by protein engineering and crystallographic studies.

Abstract: The flavoenzyme ferredoxin-NADP+ reductase (FNR) catalyzes the production of NADPH during photosynthesis. Whereas the structures of FNRs from spinach leaf and a cyanobacterium as well as many of their homologs have been solved, none of these studies has yielded a productive geometry of the flavin-nicotinamide interaction. Here, we show that this failure occurs because nicotinamide binding to wild type FNR involves the energetically unfavorable displacement of the C-terminal Tyr side chain. We used mutants of this residue (Tyr 308) of pea FNR to obtain the structures of productive NADP+ and NADPH complexes. These structures reveal a unique NADP+ binding mode in which the nicotinamide ring is not parallel to the flavin isoalloxazine ring, but lies against it at an angle of approximately 30 degrees, with the C4 atom 3 A from the flavin N5 atom.

Rare codon content affects the solubility of recombinant proteins in a codon bias-adjusted Escherichia coli strain

Abstract: The expression of heterologous proteins in Escherichia coli is strongly affected by codon bias. This phenomenon occurs when the codon usage of the mRNA coding for the foreign protein differs from that of the bacterium. The ribosome pauses upon encountering a rare codon and may detach from the mRNA, thereby the yield of protein expression is reduced. Several bacterial strains have been engineered to overcome this effect. However, the increased rate of translation may lead to protein misfolding and insolubilization. In order to prove this assumption, the solubility of several recombinant proteins from plants was studied in a codon bias-adjusted E. coli strain. The expression of eight plant proteins in Escherichia coli BL21(DE3)-pLysS and BL21(DE3)-CodonPlus-pRIL was systematically studied. The CodonPlus strain contains extra copies of the argU, ileY, and leuW tRNA genes, which encode tRNAs that recognize the codons AGA/AGG, AUA and CUA, respectively (RIL codons). The level of expression and solubility of the recombinant proteins were analyzed by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting. We found that for all proteins the solubility was at least 25% in the BL21(DE3)-pLysS strain. However, when expressed in the BL21(DE3)-CodonPlus-pRIL strain, proteins having more than 5% of amino acids coded by RIL codons were localized mainly in the insoluble fraction. Also, their expression caused retarded growth and low cell yield in the codon bias-adjusted strain at all temperatures tested. On the contrary, the solubility of proteins containing less than 5% of amino acids coded by RIL codons remained unchanged in both strains and their expression caused no effect on cell growth. Our results show that the expression of heterologous proteins coded by high RIL codon content coding sequences in a codon bias-adjusted strain is detrimental for their solubility. Our data support the hypothesis that the possible elimination of translational pauses that increase translation rate leads to protein misfolding and aggregation. This stresses the importance of strain selection according to codon content in any scheme where a large amount of biologically active product is desirable.

Structure at 2.8 A resolution of F1-ATPase from bovine heart mitochondria.

Abstract: In the crystal structure of bovine mitochondrial F1-ATPase determined at 2.8 A resolution, the three catalytic beta-subunits differ in conformation and in the bound nucleotide. The structure supports a catalytic mechanism in intact ATP synthase in which the three catalytic subunits are in different states of the catalytic cycle at any instant. Interconversion of the states may be achieved by rotation of the alpha 3 beta 3 subassembly relative to an alpha-helical domain of the gamma-subunit.

Molecular mechanisms of photosynthesis

Abstract: 1. Light and Energy. 2. Organization and Structure of Photosynthetic Systems. 3. History and Development of Photosynthesis. 4. Photosynthetic Pigments-Structure and Spectroscopy. 5. Antenna Complexes and Energy Transfer Processes. 6. Reaction Center Complexes. 7. Electron Transfer Pathways and Components. 8. Chemiosmotic Coupling and ATP Synthesis. 9. Carbon Metabolism. 10. Genetics, Assembly and Regulation of Photosynthetic. Systems. 11. Origin and Evolution of Photosynthesis. Appendix 1. Light, Energy and Kinetics

Recombinant protein expression in Escherichia coli: advances and challenges.

Abstract: Escherichia coli is the organism of choice for the production of recombinant proteins. Its use as a cell factory is well-established and it has become the most popular expression platform. For this reason, there are many molecular tools and protocols at hand for the high-level production of recombinant proteins, such as a vast catalog of expression plasmids, a great number of engineered strains and many cultivation strategies. We review the different approaches for the synthesis of recombinant proteins in E. coli and discuss recent progress in this ever-growing field.

Targeting Antioxidants to Mitochondria by Conjugation to Lipophilic Cations

Abstract: Mitochondrial oxidative damage contributes to a range of degenerative diseases. Consequently, the selective inhibition of mitochondrial oxidative damage is a promising therapeutic strategy. One way to do this is to invent antioxidants that are selectively accumulated into mitochondria within patients. Such mitochondria-targeted antioxidants have been developed by conjugating the lipophilic triphenylphosphonium cation to an antioxidant moiety, such as ubiquinol or α-tocopherol. These compounds pass easily through all biological membranes, including the blood-brain barrier, and into muscle cells and thus reach those tissues most affected by mitochondrial oxidative damage. Furthermore, because of their positive charge they are accumulated several-hundredfold within mitochondria driven by the membrane potential, enhancing the protection of mitochondria from oxidative damage. These compounds protect mitochondria from damage following oral delivery and may therefore form the basis for mitochondria-prote...