Search

With dengue virus (DENV) becoming endemic in tropical and subtropical regions worldwide, there is a pressing global demand for effective strategies to control the mosquitoes that spread this disease. Recent advances in genetic engineering technologies have made it possible to create mosquitoes with reduced vector competence, limiting their ability to acquire and transmit pathogens. Here we describe the development of Aedes aegypti mosquitoes synthetically engineered to impede vector competence to DENV. These mosquitoes express a gene encoding an engineered single-chain variable fragment derived from a broadly neutralizing DENV human monoclonal antibody and have significantly reduced viral infection, dissemination, and transmission rates for all four major antigenically distinct DENV serotypes. Importantly, this is the first engineered approach that targets all DENV serotypes, which is crucial for effective disease suppression. These results provide a compelling route for developing effective genetic-based DENV control strategies, which could be extended to curtail other arboviruses., Author summary With limited success of traditional vector control methods to curb dengue infections and more than half of the world’s population still at risk, there is a need for novel strategies to reduce its impact on public health. Recent advances in genetic technologies has allowed for precise modifications of mosquito genome to make them resistant to infections, thus breaking the transmission cycle. Here we generated engineered Ae. aegypti mosquitoes efficiently expressing a DENV-targeting single-chain variable fragment (scFv) derived from a previously characterized broadly neutralizing human antibody, which blocked infection and transmission in these mosquitoes. To our knowledge, this is the first example of an engineered transgene capable of rendering Ae. aegypti mosquitoes 100% refractory to all four serotypes of DENV. The engineered mosquitoes, in future, could easily be paired with a gene drive, capable of spreading the transgene throughout wild disease-transmitting mosquito populations and preventing further DENV transmission. Since a number of diverse and well-characterized antibodies exist against other arboviruses (eg chikungunya and Zika, this work also provides a proof-of-concept principle for developing similar genetic strategies for reducing the impact of these arboviruses.

Carbapenems are "last resort" β-lactam antibiotics used to treat serious and life-threatening health care-associated infections caused by multidrug-resistant Gram-negative bacteria. Unfortunately, the worldwide spread of genes coding for carbapenemases among these bacteria is threatening these life-saving drugs. Metallo-β-lactamases (MβLs) are the largest family of carbapenemases. These are Zn(II)-dependent hydrolases that are active against almost all β-lactam antibiotics. Their catalytic mechanism and the features driving substrate specificity have been matter of intense debate. The active sites of MβLs are flanked by two loops, one of which, loop L3, was shown to adopt different conformations upon substrate or inhibitor binding, and thus are expected to play a role in substrate recognition. However, the sequence heterogeneity observed in this loop in different MβLs has limited the generalizations about its role. Here, we report the engineering of different loops within the scaffold of the clinically relevant carbapenemase NDM-1. We found that the loop sequence dictates its conformation in the unbound form of the enzyme, eliciting different degrees of active-site exposure. However, these structural changes have a minor impact on the substrate profile. Instead, we report that the loop conformation determines the protonation rate of key reaction intermediates accumulated during the hydrolysis of different β-lactams in all MβLs. This study demonstrates the existence of a direct link between the conformation of this loop and the mechanistic features of the enzyme, bringing to light an unexplored function of active-site loops on MβLs. Fil: Palacios, Antonela Rocio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Mojica, María F.. Case Western Reserve University; Estados Unidos Fil: Giannini, Estefanía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Taracila, Magdalena A.. Case Western Reserve University; Estados Unidos. Louis Stokes Veterans Affairs Medical Center; Estados Unidos Fil: Bethel, Christopher R.. Louis Stokes Veterans Affairs Medical Center; Estados Unidos Fil: Alzari, Pedro M.. Institut Pasteur de Paris; Francia Fil: Otero, Lisandro Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Klinke, Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Llarrull, Leticia Irene. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Bonomo, Robert A.. Case Western Reserve University; Estados Unidos Fil: Vila, Alejandro Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina

International audience; Most secreted growth factors and cytokines are functionally pleiotropic because their receptors are expressed on diverse cell types. While important for normal mammalian physiology, pleiotropy limits the efficacy of cytokines and growth factors as therapeutics. Stem cell factor (SCF) is a growth factor that acts through the c-Kit receptor tyrosine kinase to elicit hematopoietic progenitor expansion, but can be toxic when administered in vivo because it concurrently activates mast cells. We engineered a mechanism-based SCF partial agonist that impaired c-Kit dimerization, truncating downstream signaling amplitude. This SCF variant elicited biased activation of hematopoietic progenitors over mast cells in vitro and in vivo. Mouse models of SCF-mediated anaphylaxis, radioprotection, and hematopoietic expansion revealed that this SCF partial agonist retained therapeutic efficacy while exhibiting virtually no anaphylactic off-target effects. The approach of biasing cell activation by tuning signaling thresholds and outputs has applications to many dimeric receptor-ligand systems.

International audience; Proteins exist as a dynamic ensemble of interconverting substates, which defines their conformational energy landscapes. Recent work has indicated that mutations that shift the balance between conformational substates (CSs) are one of the main mechanisms by which proteins evolve new functions. In the present study, we probe this assertion by examining phenotypic protein adaptation to extreme conditions, using the allosteric tetrameric lactate dehydrogenase (LDH) from the hyperthermophilic bacterium Thermus thermophilus (Tt) as a model enzyme. In the presence of fructose 1, 6 bis-phosphate (FBP), allosteric LDHs catalyze the conversion of pyruvate to lactate with concomitant oxidation of nicotinamide adenine dinucleotide, reduced form (NADH). The catalysis involves a structural transition between a low-affinity inactive "T-state" and a high-affinity active "R-state" with bound FBP. During this structural transition, two important residues undergo changes in their side chain conformations. These are R171 and H188, which are involved in substrate and FBP binding, respectively. We designed two mutants of Tt-LDH with one ("1-Mut") and five ("5-Mut") mutations distant from the active site and characterized their catalytic, dynamical, and structural properties. In 1-Mut Tt-LDH, without FBP, the K(m)(Pyr) is reduced compared with that of the wild type, which is consistent with a complete shifting of the CS equilibrium of H188 to that observed in the R-state. By contrast, the CS populations of R171, k(cat) and protein stability are little changed. In 5-Mut Tt-LDH, without FBP, K(m)(Pyr) approaches the values it has with FBP and becomes almost temperature independent, k(cat) increases substantially, and the CS populations of R171 shift toward those of the R-state. These changes are accompanied by a decrease in protein stability at higher temperature, which is consistent with an increased flexibility at lower temperature. Together, these results show that the thermal properties of an enzyme can be strongly modified by only a few or even a single mutation, which serve to alter the equilibrium and, hence, the relative populations of functionally important native-state CSs, without changing the nature of the CSs themselves. They also provide insights into the types of mutational pathways by which protein adaptation to temperature is achieved.

The variety of recombinant protein expression systems have been developed as a resource of FVII gene expression. In the current study, the authors used a novel protein expression system based on the Iranian LizardLeishmania, a trypanosomatid protozoan as a host for expression of FVII. Plasmid containing cDNA encoding full-length human FVII was introduced into LizardLeishmaniaand positive transfectants were analyzed by SDS-PAGE and Western blot analysis. Furthermore, biological activity of purified protein was detected by PT assay. The recombinant strain harboring a construct was analyzed for expression of FVII at the mRNA and protein level. Purified rFVII was obtained and in order to confirm the purified compound was in fact rFVII. Western blot analysis was carried out. Clotting time in PT assay was reduced about 30 seconds with the purified rFVII. In Conclusion, this study has demonstrated, for the first time, thatLeishmaniacells can be used as an expression system for producing recombinant FVII.

International audience; We review the current status of expression of heterologous systems for bioenergy and bioproduction in bacteria using a model-based approach. As an aim for synthetic biology, it requires mathematical models of genetic modules that could be characterized independently of their context. This fastens the design of metabolic circuits using a combinatorial design approach, where given pathways could be optimized for maximal bioproduction, while being nontoxic for the chassis. We show how recent characterization of genetic parts, such as promoters, RBS or sRNAs could be used to fine-tune the expression of individual genes to achieve that goal. We also present lists of enzymes that are used for bioproduction, enlarging such set of biological parts.

International audience; Lantibiotics are antimicrobial peptides from the bacteriocin family, secreted by Gram-positive bacteria. These peptides differ from other bacteriocins by the presence of (methyl)lanthionine residues, which result from enzymatic modification of precursor peptides encoded by structural genes. Several groups of lantibiotics have been distinguished, the largest of which is the lacticin 481 group. This group consists of at least 16 members, including lacticin 481, streptococcin A-FF22, mutacin II, nukacin ISK-1, and salivaricins. We present the first review devoted to this lantibiotic group, knowledge of which has increased significantly within the last few years. After updating the group composition and defining the common properties of these lantibiotics, we highlight the most recent developments. The latter concern: transcriptional regulation of the lantibiotic genes; understanding the biosynthetic machinery, in particular the ability to perform in vitro prepeptide maturation; characterization of a novel type of immunity protein; and broad application possibilities. This group differs in many aspects from the best known lantibiotic group (nisin group), but shares properties with less-studied groups such as the mersacidin, cytolysin and lactocin S groups.

Summary. Background: The utility of purified coagulation factor (F)VIII for treatment of hemophilia A is limited in part by its instability following activation by thrombin, which is caused by spontaneous dissociation of the A2 domain from the activated FVIII (FVIIIa) heterotrimer. To prevent this A2 domain dissociation in FVIIIa, we previously engineered a cysteine pair (C664–C1826) in recombinant FVIII that formed a disulfide bond cross-linking the A2 domain in the heavy chain to the A3 domain in the light chain. This engineered disulfide bond resulted in a more stable FVIIIa. Aims: Here, we characterize the functional parameters of C664–C1828 FVIII and of a new disulfide bond-stabilized FVIII (C662–C1828 FVIII). Methods: In order to assess whether these FVIII variants might be good candidates for a new therapeutic agent to treat hemophilia A, we investigated a variety of functional parameters that might affect the in vivo properties of the variants, including half-life of disulfide bond-stabilized FVIII and FVIIIa and the potency of these FVIIIa molecules in the FXase complex. Results: Both disulfide bond-stabilized variants had improved affinity for von Willebrand factor (VWF). In studies of FX activation by purified FIXa and FVIIIa, C662– C1828 FVIIIa had normal activity while C664–C1826 FVIIIa had reduced activity. Both C664–C1826 FVIIIa and C662– C1828 FVIIIa were inactivated by activated protein C (APC) but the rates of inactivation were different. Conclusion: Overall, the specific location of the disulfide bridge between the A2 and A3 domains appears to affect functional properties of FVIIIa. In summary, introduction of engineered interdomain disulfides results in FVIIIa variants that resist spontaneous loss of activity while retaining susceptibility to APC proteolytic inactivation and maintaining VWF binding.

International audience; Trinucleotide repeat expansions are involved in more than two dozen neurological and developmental disorders. Conventional therapeutic approaches aimed at regulating the expression level of affected genes, which rely on drugs, oligonucleotides, and/or transgenes, have met with only limited success so far. An alternative approach is to shorten repeats to non-pathological lengths using highly specific nucleases. Here, I review early experiments using meganucleases, zinc-finger nucleases (ZFN), and transcription-activator like effector nucleases (TALENs) to contract trinucleotide repeats, and discuss the possibility of using CRISPR-Cas nucleases to the same end. Although this is a nascent field, I explore the possibility of designing nucleases and effectively delivering them in the context of gene therapy.

International audience; Proteins are dynamic systems, and understanding dynamics is critical for fully understanding protein function. Therefore, the question of whether laboratory engineering has an impact on protein dynamics is of general interest. Here, we demonstrate that two homologous, naturally evolved enzymes with high degrees of structural and functional conservation also exhibit conserved dynamics. Their similar set of slow timescale dynamics is highly restricted, consistent with evolutionary conservation of a functionally important feature. However, we also show that dynamics of a laboratory-engineered chimeric enzyme obtained by recombination of the two homologs exhibits striking difference on the millisecond timescale, despite function and high-resolution crystal structure (1.05 Å) being conserved. The laboratory-engineered chimera is thus functionally tolerant to modified dynamics on the timescale of catalytic turnover. Tolerance to dynamic variation implies that maintenance of native-like protein dynamics may not be required when engineering functional proteins.

Motivation: Antibodies are able to recognize a wide range of antigens through their complementary determining regions formed by six hypervariable loops. Predicting the 3D structure of these loops is essential for the analysis and reengineering of novel antibodies with enhanced affinity and specificity. The canonical structure model allows high accuracy prediction for five of the loops. The third loop of the heavy chain, H3, is the hardest to predict because of its diversity in structure, length and sequence composition. Results: We describe a method, based on the Random Forest automatic learning technique, to select structural templates for H3 loops among a dataset of candidates. These can be used to predict the structure of the loop with a higher accuracy than that achieved by any of the presently available methods. The method also has the advantage of being extremely fast and returning a reliable estimate of the model quality. Availability and implementation: The source code is freely available at http://www.biocomputing.it/H3Loopred/ Contact: anna.tramontano@uniroma1.it Supplementary Information: Supplementary data are available at Bioinformatics online.

International audience; Engineered protein scaffolds have received considerable attention as alternatives to antibodies in both basic and applied research, as they can offer superior biophysical properties often associated with a simpler molecular organization. Sac7d has been demonstrated as an effective scaffold for molecular recognition. Here, we used the initial L1 'flat surface' library constructed by randomization of 14 residues, to identify ligands specific for human immunoglobulin G. To challenge the plasticity of the Sac7d protein scaffold, we designed the alternative L2 'flat surface & loops' library whereof only 10 residues are randomized. Representative binders (Affitins) of the two libraries exhibited affinities in the low nanomolar range and were able to recognize different epitopes within human immunoglobulin G. These Affitins were stable up to pH 12 while largely conserving other favorable properties of Sac7d protein, such as high expression yields in Escherichia coli, solubility, thermal stability up to 80.7°C, and acidic stability (pH 0). In agreement with our library designs, mutagenesis study revealed two distinct binding areas, one including loops. Together, our results indicate that the Sac7d scaffold tolerates alternative library designs, which further expands the diversity of Affitins and may provide a general way to create tailored affinity tools for demanding applications.

International audience; The Flavivirus genus includes widespread and severe human pathogens like the four serotypes of dengue virus (DENV1 to DENV4), yellow fever virus, Japanese encephalitis virus and West Nile virus. Domain III (ED3) of the viral envelope protein interacts with cell receptors and contains epitopes recognized by virus neutralizing antibodies. Its structural, antigenic and immunogenic properties have been thoroughly studied contrary to its physico-chemical properties. Here, the ED3 domains of the above pathogenic flaviviruses were produced in the periplasm of Escherichia coli. Their thermodynamic stabilities were measured and compared in experiments of unfolding equilibriums, induced with chemicals or heat and monitored through protein fluorescence. A designed ED3 domain, with the consensus sequence of DENV strains from all serotypes, was highly stable. The low stability of the ED3 domain from DENV3 was increased by three changes of residues in the protein core without affecting its reactivity towards DENV-infected human serums. Additional changes showed that the stability of ED3 varied with the DENV3 genotype. The T(m) of ED3 was higher than 69°C for all the tested viruses and reached 86°C for the consensus ED3. The latter, deprived of its disulfide bond by mutations, was predominantly unfolded at 20°C. These results will help better understand and design the properties of ED3 for its use as diagnostic, vaccine or therapeutic tools.

International audience; Fibrillar α-synuclein (α-Syn) is the principal component of Lewy bodies, which are evident in individuals affected by Parkinson disease (PD). This neuropathologic form of α-Syn plays a central role in PD progression as it has been shown to propagate between neurons. Tools that interfere with α-Syn assembly or change the physicochemical properties of the fibrils have potential therapeutic properties as they may be sufficient to interfere with and/or halt cell-to-cell transmission and the systematic spread of α-Syn assemblies within the central nervous system. Vertebrate molecular chaperones from the constitutive/heat-inducible heat shock protein 70 (Hsc/p70) family have been shown to hinder the assembly of soluble α-Syn into fibrils and to bind to the fibrils and very significantly reduce their toxicity. To understand how Hsc70 family members sequester soluble α-Syn, we set up experiments to identify the molecular chaperone-α-Syn surface interfaces. We cross-linked human Hsc70 and its yeast homologue Ssa1p and α-Syn using a chemical cross-linker and mapped the Hsc70- and Ssa1p-α-Syn interface. We show that the client binding domain of Hsc70 and Ssa1p binds two regions within α-Syn similar to a tweezer, with the first spanning residues 10-45 and the second spanning residues 97-102. Our findings define what is necessary and sufficient for engineering Hsc70- and Ssa1p-derived polypeptide with minichaperone properties with a potential as therapeutic agents in Parkinson disease through their ability to affect α-Syn assembly and/or toxicity.

International audience; Mitochondrial aminoacyl-tRNA synthetases are key enzymes in translation. They are encoded by the nuclear genome, synthesized as precursors in the cytosol and imported. Most are matured by cleavage of their N-terminal targeting sequence. The poor expression of mature proteins in prokaryotic systems, along with their low solubility and stability after purification are major obstacles for biophysical and crystallographic studies. The purpose of the present work was to analyze the influence of additives on a slightly soluble aspartyl-tRNA synthetase and of the N-terminal sequence of the protein on its expression and solubility. On the one hand, the solubility of the enzyme was augmented to some extent in the presence of a chemical analog of the intermediary product aspartyl-adenylate, 5'-O-[N-(L aspartyl) sulfamoyl] adenosine. On the other hand, expression was enhanced by extending the N-terminus by seven natural amino acids from the predicted targeting sequence. The re-designed enzyme was active, monodisperse, more soluble and yielded crystals that are suitable for structure determination. This result underlines the importance of the N-terminal residue sequence for solubility. It suggests that additional criteria should be taken into account for the prediction of cleavage sites in mitochondrial targeting sequences.

The protein folding problem is often studied by comparing the mechanisms of proteins sharing the same structure but different sequence. The recent design of the two proteins G(A)88 and G(B)88, displaying different structures and functions while sharing 88% sequence identity (49 out of 56 amino acids), allows the unique opportunity for a complementary approach. At which stage of its folding pathway does a protein commit to a given topology? Which residues are crucial in directing folding mechanisms to a given structure? By using a combination of biophysical and computational techniques, we have characterized the folding of both G(A)88 and G(B)88. We show that, contrary to expectation, G(B)88, characterized by a native α+β fold, displays in the denatured state a content of native-like helical structure greater than G(A)88, which is all-α in its native state. Both experiments and simulations indicate that such residual structure may be tuned by changing pH. Thus, despite the high sequence identity, the folding pathways for these two proteins appear to diverge as early as in the denatured state. Our results suggest a mechanism whereby protein topology is committed very early along the folding pathway, being imprinted in the residual structure of the denatured state.

Chitosan oligosaccharides (oligomers of (GlcNAc) x (GlcN) y ) are used in the pharmaceutical, cosmetic and food industries and are reported to have therapeutic benefits. However, it is unknown whether their biological activity depends on the degree of deacetylation or the sequence of residues within the oligomer. We report here the development of a random mutagenesis method for directed evolution of Streptomyces lividans acetyl xylan esterase (AxeA), which we previously showed is able to deacetylate chitinous substrate, in order to obtain chitooligosaccharides with well-defined structural properties. A colorimetric assay was used to pre-screen libraries for p -nitrophenol acetate hydrolysis activity and an HPLC-UV absorbance assay was optimized to subsequently screen for deacetylase activity toward hexa- N -acetyl-glucosamine substrate (GlcNAc) 6 . Native AxeA and two variants displaying > 50% deacetylation of the oligohexamer substrate after reaction at 50 °C for 24 h in diluted culture supernatant were then selected for detailed analysis of the enzymatic products. A HILIC (hydrophilic interaction chromatography)-mode LC method was developed for profiling the deacetylated chitooligosaccharide products and HILIC-MS/MS sequencing revealed that ca. 30 different deacetylation products ranging from (GlcNAc) 5 (GlcN) 1 to (GlcNAc) 1 (GlcN) 5 and isomers thereof were produced. The AxeA variants produced, on average, 26% more unique products than the native enzyme; however, none were able to fully deacetylate the substrate to make (GlcN) 6 . The long term goal of this multidisciplinary approach is to improve the activity of chitosan oligosaccharides to an industrially applicable level.

International audience; Cilia and flagella are evolutionary conserved organelles that generate fluid movement and locomotion, and play roles in chemosensation, mechanosensation and intracellular signalling. In complex organisms, cilia are highly diversified, which allows them to perform various functions; however, they retain a 9+0 or 9+2 microtubules structure connected to a basal body. Here, we describe FOR20 (FOP-related protein of 20 kDa), a previously uncharacterized and highly conserved protein that is required for normal formation of a primary cilium. FOR20 is found in PCM1-enriched pericentriolar satellites and centrosomes. FOR20 contains a Lis1-homology domain that promotes self-interaction and is required for its satellite localization. Inhibition of FOR20 expression in RPE1 cells decreases the percentage of ciliated cells and the length of the cilium on ciliated cells. It also modifies satellite distribution, as judged by PCM1 staining, and displaces PCM1 from a detergent-insoluble to a detergent-soluble fraction. The subcellular distribution of satellites is dependent on both microtubule integrity and molecular motor activities. Our results suggest that FOR20 could be involved in regulating the interaction of PCM1 satellites with microtubules and motors. The role of FOR20 in primary cilium formation could therefore be linked to its function in regulating pericentriolar satellites. A role for FOR20 at the basal body itself is also discussed.

International audience; Tissue plasminogen activator (t-PA) is one of the fibrin-specific serine proteases that play a crucial role in the fibrinolytic system. The rapid clearance of the drug from the circulation, caused by its active uptake in the liver, has lead to complicated clinical applications. Different forms of plasminogen activators have been developed to treat thrombotic disease. Deletion of the first three domains of t-PA by gene manipulation techniques has shown a significant increase in its plasma half life. In order to compensate the disadvantage of higher bleeding risk, a novel chimeric truncated form of t-PA with 394 amino acids and more fibrin affinity compared to the truncated form was designed to be expressed in Chinese Hamster Ovarian (CHO) cells. The recombinant chimeric plasminogen activator consists of kringle 2 and serine protease (K2S) domains of t-PA, namely GHRP-SYQ-K2S. The level of expression was found to be 752 IU/ml with 566,917 IU/mg specific activity, based on amidolytic activity. The fibrin binding of this novel chimeric truncated t-PA was 86% of the full length t-PA at a fibrinogen concentration of 0.2 mg/ml. This could be a promising approach with more desirable pharmacodynamic properties compared to existing commercial forms.

International audience; Protein design has many applications not only in biotechnology but also in basic science. It uses our current knowledge in structural biology to predict, by computer simulations, an amino acid sequence that would produce a protein with targeted properties. As in other examples of synthetic biology, this approach allows the testing of many hypotheses in biology. The recent development of automated computational methods to design proteins has enabled proteins to be designed that are very different from any known ones. Moreover, some of those methods mostly rely on a physical description of atomic interactions, which allows the designed sequences not to be biased towards known proteins. In this paper, we will describe the use of energy functions in computational protein design, the use of atomic models to evaluate the free energy in the unfolded and folded states, the exploration and optimization of amino acid sequences, the problem of negative design and the design of biomolecular function. We will also consider its use together with the experimental techniques such as directed evolution. We will end by discussing the challenges ahead in computational protein design and some of their future applications.

Protein aggregation is a main barrier hindering structural and functional studies of a number of interesting biological targets. The E6 oncoprotein of Human Papillomavirus strain 16 (E6(16)) is difficult to express under a native soluble form in bacteria. Produced as an unfused sequence, it forms inclusion bodies. Fused to the C-terminus of MBP, it is mainly produced in the form of soluble high molecular weight aggregates. Here, we produced as MBP-fusions seven E6 proteins from other HPV strains (5, 11, 18, 33, 45, 52, and 58) belonging to four different species, and we compared their aggregation state to that of MBP-E6(16). Using a fast mutagenesis method, we changed most non-conserved cysteines to the isosteric residue serine to minimize disulfide bridge-mediated aggregation during purification. Static and dynamic light scattering measurements, ultracentrifugation and electron microscopy demonstrated the presence in all MBP-E6 preparations of soluble high-molecular weight aggregates with a well-defined spherical shape. These aggregated particles are relatively monodisperse but their amount and their size vary depending on the conditions of expression and the strain considered. For all strains, minimal aggregate formation occurs when the expression is performed at 15 degrees C. Such observations suggest that the assembly of MBP-E6 aggregates takes place in vivo during protein biosynthesis, rather than occurring during purification. Finally, we show that all MBP-E6 preparations contain two zinc ions per protein monomer, suggesting that E6 domains within the high molecular weight aggregates possess a native-like fold, which enables correct coordination to the metal center.

International audience; Recombinant rabies virus glycoprotein (rRVGP) was expressed in Drosophila melanogaster Schneider 2 (S2) cells. The cDNA encoding the entire RVGP gene was cloned in an expression plasmid under the control of the constitutive actin promoter (Ac), which was co-transfected into S2 cells together with a hygromycin selection plasmid. Selected S2 cell populations (S2AcRVGP) had a decreased ability to grow and consume substrates, when compared to the non-transfected cells (S2). They were shown, by PCR, to express the RVGP gene and mRNA and, by immunoblotting, to synthesize the rRVGP in its expected molecular mass of 65 kDa. ELISA kinetic studies showed the rRVGP expression in cell lysates and supernatants attaining concentrations of 300 microg/L. By flow cytometry analysis, about 30% of the cells in the co-transfected populations were shown to express the rRVGP. Cell populations selected by limiting dilution expressed higher rRVGP yields. Mice immunized with rRVGP were shown to synthesize antibodies against rabies virus and be protected against experimental infection with rabies virus. The data presented here show that S2 cells can be suitable hosts for the rRVGP expression, allowing its synthesis in a high degree of physical and biological integrity.

International audience; We report here a targeting method that exploits the expression pattern of cell surface proteases to induce gene delivery to specific tissues. We describe retroviral vectors harboring modified surface glycoproteins derived from an avian influenza virus hemagglutinin (HA) for which the cell entry properties, dependent on HA cleavage by producer cells, were conditionally blocked at a postbinding step by insertion of matrix metalloproteinase (MMP) substrates. We demonstrate that such vectors induce gene transfer, both in vitro and in mice harboring human tumor xenografts, only through contact with target cells expressing MMPs that cleave the substrate introduced into the recombinant HA. This selective gene transfer in MMP-rich cells was specifically inhibited by 1,10-phenanthroline, a broad-range MMP inhibitor. Importantly, such MMP-activatable vectors selectively transduced MMP-rich cells in heterogeneous populations containing MMP-rich and MMP-poor cells. These vectors will allow useful gene transfer applications into target cells exhibiting specific protease activities.

Laccases are oxidizing enzymes of interest because of their potential environmental and industrial applications. We performed site-directed mutagenesis of a laccase produced by Trametes versicolor in order to improve its catalytic properties. Considering a strong interaction of the Asp residue in position 206 with the substrate xylidine, we replaced it with Glu, Ala or Asn, expressed the mutant enzymes in the yeast Yarrowia lipolytica and assayed the transformation of phenolic and non-phenolic substrates. The transformation rates remain within the same range whatever the mutation of the laccase and the type of substrate: at most a 3-fold factor increase was obtained for k(cat) between the wild-type and the most efficient mutant Asp206Ala with 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic) acid as a substrate. Nevertheless, the Asn mutation led to a significant shift of the pH (DeltapH = 1.4) for optimal activity against 2,6-dimethoxyphenol. This study also provides a new insight into the binding of the reducing substrate into the active T1 site and induced modifications in catalytic properties of the enzyme.

Neurons regulate the propagation of chemoelectric signals throughout the nervous system by opening and closing ion channels, a process known as gating. Here, histidine-based metal-binding sites were engineered along the intrinsic pore of a chimeric Cys-loop receptor to probe state-dependent Zn 2+ -channel interactions. Patterns of Zn 2+ ion binding within the pore reveal that, in the closed state, the five pore-lining segments adopt an oblique orientation relative to the axis of ion conduction and constrict into a physical gate at their intracellular end. The interactions of Zn 2+ with the open state indicate that the five pore-lining segments should rigidly tilt to enable the movement of their intracellular ends away from the axis of ion conduction, so as to open the constriction (i.e., the gate). Alignment of the functional results with the 3D structure of an acetylcholine receptor allowed us to generate structural models accounting for the closed and open pore conformations and for a gating mechanism of a Cys-loop receptor.

The difficulty and expense of preparing protein samples highly enriched in stable isotopes is a bottleneck for structural studies by nuclear magnetic resonance (NMR) spectroscopy. We have developed a new regulatable expression/labeling vector system in the cyanobacterium Anabaena sp. PCC 7120 using the endogenous promoter of the nitrate assimilation nir operon. Standard proteins were overexpressed upon induction with NaNO3, yielding up to 250 mg/L of culture. When the cyanobacteria were grown in the presence of inexpensive 15N-, 13C-labeled mineral salts and 2H2O, the expressed polypeptides were highly (>90%) enriched in stable isotopes. Furthermore, the tight repression of the nir promoter upon induction allowed the production of the toxic oncoprotein E6. In addition, under these conditions, the malE31 protein, while insoluble in Escherichia coli, was found to be soluble in Anabaena. Together, these properties render the described system especially suitable for the production and/or triple labeling of recombinant protein samples. It represents an interesting alternative to conventional protein expression systems used in structural genomics.

International audience; Enzyme engineering by molecular modelling and site-directed mutagenesis can be remarkably efficient. Directed enzyme evolution appears as a more general strategy for the isolation of catalysts as it can be applied to most chemical reactions in aqueous solutions. Selections, as opposed to screening, allow the simultaneous analysis of protein properties for sets of up to about 10(14) different proteins. These approaches for the parallel processing of molecular information 'Is the protein a catalyst?' are reviewed here in the case of selections based on the formation of a specific reaction product. Several questions are addressed about in vivo and in vitro selections for catalysis reported in the literature. Can the selection system be extended to other types of enzymes? Does the selection control regio- and stereo-selectivity? Does the selection allow the isolation of enzymes with an efficient turnover? How should substrates be substituted or mimicked for the design of efficient selections while minimising the number of chemical synthesis steps? Engineering sections provide also some clues to design selections or to circumvent selection biases. A special emphasis is put on the comparison of in vivo and in vitro selections for catalysis.

Predictive engineering of antibodies exhibiting fast kinetic properties could provide reagents for biotechnological applications such as continuous monitoring of compounds or affinity chromatography. Based on covariance analysis of murine germline antibody variable domains, we selected position L34 (Kabat numbering) for mutational studies. This position is located at the VL/VH interface, at the base of the paratope but with limited antigen contacts, thus making it an attractive position for mild alterations of antigen binding properties. We introduced a serine at position L34 in two different antibodies: Fab (fragment antigen binding) 57P (Asn34Ser) and scFv (single chain fragment variable) 1F4 (Gln34Ser), that recognize peptides derived from the coat protein of tobacco mosaic virus and the oncoprotein E6, respectively. Both mutated antibodies exhibited similar properties: (i) expression levels of active fragments in Escherichia coli were markedly improved; (ii) thermostability was enhanced; and (iii) dissociation rate parameters (k(off)) were increased by 2- and at least 57-fold for scFv1F4 and Fab57P, respectively, while their association rate parameters (k(on)) remained unchanged. The L34 Ala and Thr mutants of both antibody fragments did not possess these properties. This first demontration of similar effects observed in two antibodies with different specificities may open the way to the predictive design of molecules with enhanced stability and fast dissociation rates.

International audience; Several proteins of Clostridium thermocellum possess a C-terminal triplicated sequence related to bacterial cell surface proteins. This sequence was named the SLH domain (for S-layer homology), and it was proposed that it might serve to anchor proteins to the cell surface (A. Lupas, H. Engelhardt, J. Peters, U. Santarius, S. Volker, and W. Baumeister, J. Bacteriol. 176:1224–1233, 1994). This hypothesis was investigated by using the SLH-containing protein ORF1p from C. thermocellum as a model. Subcellular fractionation, immunoblotting, and electron microscopy of immunocytochemically labeled cells indicated that ORF1p was located on the surface of C. thermocellum. To detect C. thermocellum components interacting with the SLH domains of ORF1p, a probe was constructed by grafting these domains on the C terminus of the MalE protein of Escherichia coli. The SLH domains conferred on the chimeric protein (MalE-ORF1p-C) the ability to bind noncovalently to the peptidoglycan of C. thermocellum. In addition, 125 I-labeled MalE-ORF1p-C was shown to bind to SLH-bearing proteins transferred onto nitrocellulose, and to a 26-to 28-kDa component of the cell envelope. These results agree with the hypothesis that SLH domains contribute to the binding of exocellular proteins to the cell surface of bacteria. The gene carrying ORF1 and its product, ORF1p, are renamed olpB and OlpB (for outer layer protein B), respectively.