Publications

2008

• Binding of tetracyclines to elongation factor Tu, the Tet repressor, and the ribosome: a molecular dynamics simulation study.
  
  • Simonson Thomas
  • Aleksandrov Alexey
  Biochemistry, American Chemical Society, 2008, 47 (51), pp.13594-603. Tetracycline (Tc) is a broad-spectrum antibiotic that kills bacteria by interrupting protein biosynthesis. It is thought that the bacteriostatic action of Tc is associated with its binding to the acceptor site (or A site) in the bacterial ribosome, interfering with the attachment of aminoacyl-tRNA. Recently, however, the crystal structure of a complex between Tc and trypsin-modified elongation factor Tu (tm-EF-Tu) was determined, raising the question of whether Tc binding to EF-Tu has a role in its inhibition of protein synthesis. We address this question using computer simulations. As controls, we first compute relative ribosome binding free energies for seven Tc variants for which experimental data are available, obtaining good agreement. We then consider the binding of Tc to both the trypsin-modified and unmodified EF-Tu-GDP complexes. We show that the direct contribution of EF-Tu to the binding free energy is negligible; rather, the binding can be solely attributed to interactions of Tc with a bridging Mg(2+) ion and the GDP phosphate groups. The effects of trypsin modification are modest. Further, our calculations show that EF-Tu does not exhibit any binding preference for Tc over the nonantibiotic, 4-dedimethyl-Tc, and EF-Tu does not bind the Tc analogue tigecycline, which is a potent antibiotic. In contrast, both the ribosome and the Tet Repressor protein (involved in Tc resistance) do show a binding preference for Tc over 4-dedimethyl-Tc, and the ribosome prefers to bind tigecycline over Tc. Overall, our results provide insights into the binding properties of tetracyclines and support the idea that EF-Tu is not their primary target. (10.1021/bi801726q)
  DOI : 10.1021/bi801726q
• Pareto optimization in computational protein design with multiple objectives.
  
  • Suarez Maria
  • Tortosa Pablo
  • Carrera J.
  • Jaramillo A.
  Journal of Computational Chemistry, Wiley, 2008, 29 (16), pp.2704-11. The optimization for function in computational design requires the treatment of, often competing, multiple objectives. Current algorithms reduce the problem to a single objective optimization problem, with the consequent loss of relevant solutions. We present a procedure, based on a variant of a Pareto algorithm, to optimize various competing objectives in protein design that allows reducing in several orders of magnitude the search of the solution space. Our methodology maintains the diversity of solutions and provides an iterative way to incorporate automatic design methods in the design of functional proteins. We have applied our systematic procedure to design enzymes optimized for both catalysis and stability. However, this methodology can be applied to any computational chemistry application requiring multi-objective combinatorial optimization techniques. (10.1002/jcc.20981)
  DOI : 10.1002/jcc.20981
• PROTDES: CHARMM toolbox for computational protein design.
  
  • Suarez Maria
  • Tortosa Pablo
  • Jaramillo Alfonso
  Systems and Synthetic Biology, Springer Verlag (Germany), 2008, 2 (3-4), pp.105-13. We present an open-source software able to automatically mutate any residue positions and find the best aminoacids in an arbitrary protein structure without requiring pairwise approximations. Our software, PROTDES, is based on CHARMM and it searches automatically for mutations optimizing a protein folding free energy. PROTDES allows the integration of molecular dynamics within the protein design. We have implemented an heuristic optimization algorithm that iteratively searches the best aminoacids and their conformations for an arbitrary set of positions within a structure. Our software allows CHARMM users to perform protein design calculations and to create their own procedures for protein design using their own energy functions. We show this by implementing three different energy functions based on different solvent treatments: surface area accessibility, generalized Born using molecular volume and an effective energy function. PROTDES, a tutorial, parameter sets, configuration tools and examples are freely available at http://soft.synth-bio.org/protdes.html . (10.1007/s11693-009-9026-7)
  DOI : 10.1007/s11693-009-9026-7
• Expression and characterization of a computationally designed laccase-like enzyme
  
  • Glykys D. J.
  • Szilvay G.
  • Tortosa Pablo
  • Suarez Maria
  • Jaramillo Alfonso
  , 2008, pp.618c. Protein engineering of oxido-reductase enzymes, such as laccases, has attracted considerable interest due to their present and potential use in industrial applications. In an effort to improve the stability of laccases for various applications, computational methods were used to design a laccase-like enzyme with optimized folding characteristics and improved stability. The laccase glycoprotein from the fungus Trametes versicolor (TvL) was used as a basis for designing a new laccase-like protein. The backbone structure of TvL was left intact while all the amino acids except for the ones around the trinuclear copper center were optimized for stability by computational methods. This newly designed laccase-like ~60 kDa amino acid chain was optimized for E. coli expression and was inserted in a pET vector with a C-terminal poly-His tag. Since this enzyme is expressed in bacteria, it will be non-glycosylated. This laccase-like enzyme has been successfully expressed in the presence of copper following IPTG induction in E. coli. The majority of the expressed protein was found in the inclusion body fraction. In order to refold the protein, different strategies of denaturation and folding have been utilized. Under the right conditions, a small amount of the protein can be found in the soluble fraction and it has been purified using metal affinity, ion exchange and gel filtration chromatography methods. The characterization of the final protein product includes molecular weight spectra (MALDI-TOF), conformational analysis (CD), thermal stability (CD) and copper content. Lastly, the kinetic activity of the enzyme under several conditions and with various substrates will be determined and compared to the original TvL laccase as well as to previously studied laccases from other sources.
• Neutral evolution of proteins: The superfunnel in sequence space and its relation to mutational robustness.
  
  • Noirel Josselin
  • Simonson Thomas
  The Journal of Chemical Physics, American Institute of Physics, 2008, 129 (18), pp.185104. Following Kimura's neutral theory of molecular evolution [M. Kimura, The Neutral Theory of Molecular Evolution (Cambridge University Press, Cambridge, 1983) (reprinted in 1986)], it has become common to assume that the vast majority of viable mutations of a gene confer little or no functional advantage. Yet, in silico models of protein evolution have shown that mutational robustness of sequences could be selected for, even in the context of neutral evolution. The evolution of a biological population can be seen as a diffusion on the network of viable sequences. This network is called a "neutral network." Depending on the mutation rate mu and the population size N, the biological population can evolve purely randomly (muN<1) or it can evolve in such a way as to select for sequences of higher mutational robustness (muN>1). The stringency of the selection depends not only on the product muN but also on the exact topology of the neutral network, the special arrangement of which was named "superfunnel." Even though the relation between mutation rate, population size, and selection was thoroughly investigated, a study of the salient topological features of the superfunnel that could affect the strength of the selection was wanting. This question is addressed in this study. We use two different models of proteins: on lattice and off lattice. We compare neutral networks computed using these models to random networks. From this, we identify two important factors of the topology that determine the stringency of the selection for mutationally robust sequences. First, the presence of highly connected nodes ("hubs") in the network increases the selection for mutationally robust sequences. Second, the stringency of the selection increases when the correlation between a sequence's mutational robustness and its neighbors' increases. The latter finding relates a global characteristic of the neutral network to a local one, which is attainable through experiments or molecular modeling. (10.1063/1.2992853)
  DOI : 10.1063/1.2992853
• DESHARKY: automatic design of metabolic pathways for optimal cell growth.
  
  • Rodrigo G.
  • Carrera J.
  • Prather K.J.
  • Jaramillo A.
  Bioinformatics, Oxford University Press (OUP), 2008, 24 (21), pp.2554-6. MOTIVATION: The biological solution for synthesis or remediation of organic compounds using living organisms, particularly bacteria and yeast, has been promoted because of the cost reduction with respect to the non-living chemical approach. In that way, computational frameworks can profit from the previous knowledge stored in large databases of compounds, enzymes and reactions. In addition, the cell behavior can be studied by modeling the cellular context. RESULTS: We have implemented a Monte Carlo algorithm (DESHARKY) that finds a metabolic pathway from a target compound by exploring a database of enzymatic reactions. DESHARKY outputs a biochemical route to the host metabolism together with its impact in the cellular context by using mathematical models of the cell resources and metabolism. Furthermore, we provide the sequence of amino acids for the enzymes involved in the route closest phylogenetically to the considered organism. We provide examples of designed metabolic pathways with their genetic load characterizations. Here, we have used Escherichia coli as host organism. In addition, our bioinformatic tool can be applied for biodegradation or biosynthesis and its performance scales with the database size. AVAILABILITY: Software, a tutorial and examples are freely available and open source at http://soft.synth-bio.org/desharky.html (10.1093/bioinformatics/btn471)
  DOI : 10.1093/bioinformatics/btn471
• Nanobiosensors based on functionalised silicon nanowires
  
  • Lehoucq G.
  • Mugherli Laurent
  • Baraton L.
  • Fromant Michel
  • Gowtham M.
  • Bondavalli P.
  • Plateau Pierre
  • Legagneux P.
  • Blanquet Sylvain
  • Charlot S.
  • Gué Anne Marie
  • Pribat D.
  , 2008.
• Structure of the archaeal Kae1/Bud32 fusion protein MJ1130: a model for the eukaryotic EKC/KEOPS subcomplex.
  
  • Hecker Arnaud
  • Lopreiato Raffaele
  • Graille Marc
  • Collinet Bruno
  • Forterre Patrick
  • Libri Domenico
  • van Tilbeurgh Herman
  EMBO Journal, EMBO Press, 2008, 27 (17), pp.2340-51. The EKC/KEOPS yeast complex is involved in telomere maintenance and transcription. The Bud32p and kinase-associated endopeptidase 1 (Kaelp) components of the complex are totally conserved in eukarya and archaea. Their genes are fused in several archaeal genomes, suggesting that they physically interact. We report here the structure of the Methanocaldococcus jannaschii Kae1/Bud32 fusion protein MJ1130. Kae1 is an iron protein with an ASKHA fold and Bud32 is an atypical small RIO-type kinase. The structure MJ1130 suggests that association with Kae1 maintains the Bud32 kinase in an inactive state. We indeed show that yeast Kae1p represses the kinase activity of yeast Bud32p. Extensive conserved interactions between MjKae1 and MjBud32 suggest that Kae1p and Bud32p directly interact in both yeast and archaea. Mutations that disrupt the Kae1p/Bud32p interaction in the context of the yeast complex have dramatic effects in vivo and in vitro, similar to those observed with deletion mutations of the respective components. Direct interaction between Kae1p and Bud32p in yeast is required both for the transcription and the telomere homeostasis function of EKC/KEOPS. (10.1038/emboj.2008.157)
  DOI : 10.1038/emboj.2008.157
• A unique conformation of the anticodon stem-loop is associated with the capacity of tRNAfMet to initiate protein synthesis.
  
  • Barraud Pierre
  • Schmitt Emmanuelle
  • Mechulam Yves
  • Dardel Frédéric
  • Tisné Carine
  Nucleic Acids Research, Oxford University Press, 2008, 36 (15), pp.4894-901. In all organisms, translational initiation takes place on the small ribosomal subunit and two classes of methionine tRNA are present. The initiator is used exclusively for initiation of protein synthesis while the elongator is used for inserting methionine internally in the nascent polypeptide chain. The crystal structure of Escherichia coli initiator tRNA(f)(Met) has been solved at 3.1 A resolution. The anticodon region is well-defined and reveals a unique structure, which has not been described in any other tRNA. It encompasses a Cm32*A38 base pair with a peculiar geometry extending the anticodon helix, a base triple between A37 and the G29-C41 pair in the major groove of the anticodon stem and a modified stacking organization of the anticodon loop. This conformation is associated with the three GC basepairs in the anticodon stem, characteristic of initiator tRNAs and suggests a mechanism by which the translation initiation machinery could discriminate the initiator tRNA from all other tRNAs. (10.1093/nar/gkn462)
  DOI : 10.1093/nar/gkn462
• Dielectric relaxation in proteins: the computational perspective.
  
  • Simonson Thomas
  Photosynthesis Research, Springer Verlag, 2008, 97 (1), pp.21-32. In photoexcitation and electron transfer, a new dipole or charge is introduced, and the structure is adjusted. This adjustment represents dielectric relaxation, which is the focus of this review. We concentrate on a few selected topics. We discuss linear response theory, as a unifying framework and a tool to describe non-equilibrium states. We review recent, molecular dynamics simulation studies that illustrate the calculation of dynamic and thermodynamic properties, such as Stokes shifts or reorganization free energies. We then turn to the macroscopic, continuum electrostatic view. We recall the physical definition of a dielectric constant and revisit the decomposition of the free energy into a reorganization and a static term. We review some illustrative continuum studies and discuss some difficulties that can arise with the continuum approach. In conclusion, we consider recent developments that will increase the accuracy and broaden the scope of all these methods. (10.1007/s11120-008-9293-2)
  DOI : 10.1007/s11120-008-9293-2
• Protéines : Structure fonction et évolution.
  
  • Aleksandrov Alexey
  , 2008. Tétracyclines (TC) sont une famille d'antibiotiques important, qui se lient SPECI ャ ... allié aux protéines du ribosome et solidaire. Le mécanisme le plus important de la résistance à la tétracycline est régie par sa reliure en Tc: Mg2 + complexe à la protéine Tet Repressor (TetR). Il est donc d'intérêt pour améliorer notre compréhension des deux Tc: TetR et Tc: liaison au ribosome. Les structures cristallines des tétracyclines dans plusieurs complexes avec les protéines et les ribosomes ont fourni des informations essentielles. Une approche complémentaire consiste à développer des modèles de simulation par ordinateur, qui peut être utilisée pour étudier la structure, la dynamique et la thermodynamique de Tc: protéines ou Tc: complexes ribosome. Malgré son importance, à la tétracycline a rarement été soumis à la modélisation informatique, en partie en raison de la nécessité de ャ> St développer un modèle de mécanique moléculaire pour le TC. Ici, nous avons développé un tel modèle, de manière à être compatible avec le ャ CHARMM27 force »ld pour les protéines et les acides nucléiques, de 12 analogues de tétracycline importants, notamment la tétracycline plaine. paramètres ld ャ forces intermoléculaires ont été dérivées de supermolécule une approche standard. Le modèle reproduit la géométrie ab initio et Fxibility ャ de chaque Tc. Comme les tests, nous avons fait des simulations d'un cristal de Tc, Tc: Mg2 + et Tc: complexes Ca2 + en solution aqueuse, et d'un complexe solvaté entre TC: Mg2 + et le TetR. Le modèle se compare bien avec un large corpus de données expérimentales. Nous ャ> St utilisé notre modèle pour l'étude Tc: reconnaissance TetR qui est un problème complexe. Nous avons utilisé des simulations d'énergie libre pour étudier les interactions électrostatiques entre la protéine et ligand et le rôle éventuel de ャ induite "en Tc contraignant. Nous avons constaté que la tétracycline préfère une étendue, l'état zwitterioniques fois en solution et en complexe avec la protéine. Tc est donc préorganisés pour la reliure. En l'absence de Tc, TetR est étroitement liée à son ADN opérateur; lors de la liaison de Tc il se dissocie de l'ADN, permettant l'expression des gènes réprimés. Son contrôle serré par Transports Canada fait TetR largement utilisables dans le génie génétique. Le Tc site de liaison est plus de 20 ヒ A partir de l'ADN, de sorte que le signal de liaison doivent se propager sur une longue distance. Nous utilisons des simulations de dynamique moléculaire et calculs continuum électrostatique pour élucider le mécanisme allostérique. Lorsque [TC: Mg] lie +, l'ion Mg2 + permet des interactions avec hélice 8 de TetR un monomère et Helix 6 de l'autre monomère, et Helix 6 est tiré en direction du noyau central de la structure. Hy- interactions drophobic avec hélice 6 puis tirez hélice 4 dans un mouvement pendulaire, avec un déplacement maximum à son extrémité N-terminale: l'interface de l'ADN. Le résidu N-terminal de l'hélice 4, Lys48, est très conservée dans l'ADN de liaison des protéines régulatrices de la classe TetR et fait la plus grande contribution de tout acide aminé à l'TetR: l'ADN d'énergie libre contraignant. Ainsi, les changements de conformation conduire à une réduction drastique de la TetR: la liaison d'un ADN lité ャハ, permettant TetR se détacher de l'ADN. Nous avons ensuite utilisé le modèle pour l'étude Tc liaison au ribosome et de facteur d'élongation Tu (EF-Tu). Les structures cristallines de Tc lié à la Thermus thermophilus 30S sous-unité montrer le même site Tc primaire obligatoire (appelé TET1), avec la plus forte densité d'électrons Tc, à proximité du site A-, compatible avec un rôle inhibiteur. Un site secondaire Tc-contraignant, TET5 appelé a également été observée dans les deux structures. Nous avons fait de la dynamique moléculaire (MD) des simulations de sous-unités 30S du ribosome pour caractériser Tc contraignant et aider à résoudre l'ambiguïté en ce qui concerne le nombre et la force de Tc sites de liaison. Nous avons présenté des preuves pour les lier à TET1 prédominante, indiquant que d'autres sites de liaison signalés sont plus faibles et pas très occupés à des concentrations physiologiques Tc. Récemment, la structure cristalline d'un complexe entre le facteur d'allongement Tu (EF-Tu) et Tc a été résolu, ce qui soulève la question de savoir si Tc 窭 冱 contraignant à EF-Tu a un rôle dans l'inhibition de la synthèse protéique. Nous montrons que la contribution directe de EF-Tu à l'énergie libre de Tc contraignant à l'EF-Tu: PIB: complexe Mg est négligeable, mais plutôt la liaison peut être attribuée uniquement à Tc interactions avec l'ion Mg et le phosphate PIB groupes . Nous montrons aussi que EF-Tu ne présente pas de préférence contraignant pour le TC sur la non-antibiotiques, 4-dedimethyl-Tc, et EF-Tu ne lie pas la tigécycline analogique Tc, qui est un antibiotique puissant. Globalement, nos résultats appuient l'idée que les EF-Tu n'est pas la cible principale de la tétracycline. Les articles présentés ci-dessous comprennent à la fois de calcul et les résultats expérimentaux. Tout le travail expérimental a été réalisé par Winfried Hinrichs et ses collabora-teurs. Tout le travail de calcul a été fait par moi-même. Les connaissances acquises dans ce travail et les techniques de modélisation employées doivent être d'intérêt pour l'amélioration des techniques antibiotiques Tc et TetR protéines améliorées pour la régulation des gènes.
• Computational design and evolution of the oscillatory response under light-dark cycles.
  
  • Rodrigo G.
  • Carrera J.
  • Jaramillo A.
  Biochimie, Elsevier, 2008, 90 (6), pp.888-97. Circadian clocks are biological systems behaving as oscillators even in constant dark conditions. We propose to use a new strategy based on computational design to provide evidence on the origin and evolution of molecular clocks. We design synthetic molecular clocks having a reduced number of genes and some of them showing architectures found in nature. We analyse the response of our models under diverse forcing light-dark (LD) cycles. Our methodology allows us to evolve networks in silico using various selective pressures, which we apply to the analysis of clocks evolved to be either autonomous or phase locked. Our designed networks either have an oscillatory response with the same period as the forcing LD cycle, or they maintain their free-running period. Our methodology will allow analysing the automatic creation of a free-running period under various LD forcing functions and learning new design principles for circadian clocks. (10.1016/j.biochi.2008.02.012)
  DOI : 10.1016/j.biochi.2008.02.012
• Probing electrostatic interactions and ligand binding in aspartyl-tRNA synthetase through site-directed mutagenesis and computer simulations.
  
  • Thompson Damien
  • Lazennec Christine
  • Plateau Pierre
  • Simonson Thomas
  Proteins - Structure, Function and Bioinformatics, Wiley, 2008, 71 (3), pp.1450-60. Faithful genetic code translation requires that each aminoacyl-tRNA synthetase recognise its cognate amino acid ligand specifically. Aspartyl-tRNA synthetase (AspRS) distinguishes between its negatively-charged Asp substrate and two competitors, neutral Asn and di-negative succinate, using a complex network of electrostatic interactions. Here, we used molecular dynamics simulations and site-directed mutagenesis experiments to probe these interactions further. We attempt to decrease the Asp/Asn binding free energy difference via single, double and triple mutations that reduce the net positive charge in the active site of Escherichia coli AspRS. Earlier, Glutamine 199 was changed to a negatively-charged glutamate, giving a computed reduction in Asp affinity in good agreement with experiment. Here, Lysine 198 was changed to a neutral leucine; then, Lys198 and Gln199 were mutated simultaneously. Both mutants are predicted to have reduced Asp binding and improved Asn binding, but the changes are insufficient to overcome the initial, high specificity of the native enzyme, which retains a preference for Asp. Probing the aminoacyl-adenylation reaction through pyrophosphate exchange experiments, we found no detectable activity for the mutant enzymes, indicating weaker Asp binding and/or poorer transition state stabilization. The simulations show that the mutations' effect is partly offset by proton uptake by a nearby histidine. Therefore, we performed additional simulations where the nearby Histidines 448 and 449 were mutated to neutral or negative residues: (Lys198Leu, His448Gln, His449Gln), and (Lys198Leu, His448Glu, His449Gln). This led to unexpected conformational changes and loss of active site preorganization, suggesting that the AspRS active site has a limited structural tolerance for electrostatic modifications. The data give insights into the complex electrostatic network in the AspRS active site and illustrate the difficulty in engineering charged-to-neutral changes of the preferred ligand. (10.1002/prot.21834)
  DOI : 10.1002/prot.21834
• Tet repressor induction by tetracycline: a molecular dynamics, continuum electrostatics, and crystallographic study.
  
  • Aleksandrov Alexey
  • Schuldt L.
  • Hinrichs Winfried
  • Simonson Thomas
  Journal of Molecular Biology, Elsevier, 2008, 378 (4), pp.898-912. The Tet repressor (TetR) mediates the most important mechanism of bacterial resistance against tetracycline (Tc) antibiotics. In the absence of Tc, TetR is tightly bound to its operator DNA; upon binding of Tc with an associated Mg(2+) ion, it dissociates from the DNA, allowing expression of the repressed genes. Its tight control by Tc makes TetR broadly useful in genetic engineering. The Tc binding site is over 20 A from the DNA, so the binding signal must propagate a long distance. We use molecular dynamics simulations and continuum electrostatic calculations to test two models of the allosteric mechanism. We simulate the TetR:DNA complex, the Tc-bound, "induced" TetR, and the transition pathway between them. The simulations support the model inferred previously from the crystal structures and reveal new details. When [Tc:Mg](+) binds, the Mg(2+) ion makes direct and water-mediated interactions with helix 8 of one TetR monomer and helix 6 of the other monomer, and helix 6 is pulled in towards the central core of the structure. Hydrophobic interactions with helix 6 then pull helix 4 in a pendulum motion, with a maximal displacement at its N-terminus: the DNA interface. The crystal structure of an additional TetR reported here corroborates this motion. The N-terminal residue of helix 4, Lys48, is highly conserved in DNA-binding regulatory proteins of the TetR class and makes the largest contribution of any amino acid to the TetR:DNA binding free energy. Thus, the conformational changes lead to a drastic reduction in the TetR:DNA binding affinity, allowing TetR to detach itself from the DNA. Tc plays the role of a specific Mg(2+) carrier, whereas the Mg(2+) ion itself makes key interactions that trigger the allosteric transition in the TetR:Tc complex. (10.1016/j.jmb.2008.03.022)
  DOI : 10.1016/j.jmb.2008.03.022
• Computational protein design: software implementation, parameter optimization, and performance of a simple model.
  
  • Schmidt Am Busch Marcel
  • Lopes Anne
  • Mignon David
  • Simonson Thomas
  Journal of Computational Chemistry, Wiley, 2008, 29 (7), pp.1092-102. Computational protein design will continue to improve as new implementations and parameterizations are explored. An automated protein design procedure is implemented and applied to the full redesign of 16 globular proteins. We combine established but simple ingredients: a molecular mechanics description of the protein where nonpolar hydrogens are implicit, a simple solvent model, a folded state where the backbone is fixed, and a tripeptide model of the unfolded state. Sequences are selected to optimize the folding free energy, using a simple heuristic algorithm to explore sequence and conformational space. We show that a balanced parametrization, obtained here and in our previous work, makes this procedure effective, despite the simplicity of the ingredients. Calculations were done using our Proteins @ Home distributed computing platform, with the help of several thousand volunteers. We describe the software implementation, the optimization of selected terms in the energy function, and the performance of the method. We allowed all amino acids to mutate except glycines, prolines, and cysteines. For 15 of the 16 test proteins, the scores of the computed sequences were comparable to those of natural homologues. Using the low energy computed sequences in a BLAST search of the SWISSPROT database, we could retrieve natural sequences for all protein families considered, with no high-ranking false-positives. The good stability of the designed sequences was supported by molecular dynamics simulations of selected sequences, which gave structures close to the experimental native structure. (10.1002/jcc.20870)
  DOI : 10.1002/jcc.20870
• Design of specific biochips for contrast enhancement of UV biological absorption
  
  • Robin Kristelle
  • Reverchon Jean-Luc
  • Mugherli Laurent
  • Benisty Henri
  , 2008, 6991, pp.A9912.
• Automated extraction of meaningful pathways from quantitative proteomics data.
  
  • Noirel J.
  • Ow S.Y.
  • Sanguinetti G.
  • Jaramillo A.
  • Wright P.C.
  Briefings in Functional Genomics and Proteomics, Oxford University Press, 2008, 7 (2), pp.136-46. Technological developments in the life sciences have resulted in an ever-accelerating pace of data production. Systems Biology tries to shed light upon these data by building complex models describing the interactions between biological components. However, extracting information from this morass of data requires the use of sophisticated computational techniques. Here, we propose a method suitable to integrate data drawn from quantitative proteomics into a metabolic scaffold and identify the metabolic pathways which are collectively up-regulated or down-regulated. The availability of such a tool is highly desirable as the extracted information could then be taken as a starting point for in-depth analyses, in particular in fields like Synthetic Biology, where datasets need be characterized routinely. (10.1093/bfgp/eln011)
  DOI : 10.1093/bfgp/eln011
• Molecular dynamics simulations of the 30S ribosomal subunit reveal a preferred tetracycline binding site.
  
  • Aleksandrov Alexey
  • Simonson Thomas
  Journal of the American Chemical Society, American Chemical Society, 2008, 130 (4), pp.1114-5. Tetracyclines (Tc) are important antibiotics that inhibit bacterial ribosomes. Two and six Tc binding sites, respectively, were seen in two X-ray structures of the Thermus thermophilus 30S ribosome subunit; the exact functional role of the various sites remains unclear. We study the two consensus sites, seen in both structures: a primary site, which is positioned to block tRNA A-site binding, and a secondary site, which has a weaker electron density. We combine molecular dynamics simulations and continuum electrostatic calculations to estimate the relative affinities of the two sites. The dielectric constant of the ribosome is set to 8, to reproduce the experimental binding free energy differences between Tc and its analogues minocycline and doxycycline, as well as more rigorous free energy simulations of Mg2+ binding. We find that both sites include a prebound Mg2+ ion, present before Tc binds. Using long simulations and comparing eight structural models for each site, we then show that primary site Tc binding is stronger by 1−4 kcal/mol; this range appears consistent with the crystallographically observed occupancies of the two sites. With this free energy range, TET5 is largely unoccupied under physiological conditions. Thus, we propose that the primary site is the inhibitory site and that allosteric effects may not be essential for tetracycline function. Copyright (2008) American Chemical Society.
• Evolution dirigée de deux aminoacyl-ARNt synthétases : Mise en place et applications d'une méthode de 'protein design'.
  
  • Lopes Anne
  , 2008. La conception des protéines ou ‘protein design' a pour but de développer des protéines possédant de nouvelles caractéristiques structurales et/ou fonctionnelles. Le principe consiste à identifier parmi toutes les séquences compatibles avec le repliement d'intérêt, celles qui vont conférer à la protéine, la fonction désirée. La procédure générale est réalisée en deux étapes. La première consiste à calculer une matrice d'énergie contenant les énergies d'interactions entre toutes les paires de résidus de la protéine en autorisant successivement tous les types d'acides aminés dans toutes leurs conformations possibles. La seconde étape, ou ‘phase d'optimisation', consiste à explorer simultanément l'espace des séquences et des conformations afin de déterminer la combinaison optimale d'acides aminés étant donné le repliement de départ. Ensuite, différents filtres peuvent être appliqués pour sélectionner les séquences fonctionnelles (étant donné le repliement d'intérêt) des non fonctionnelles. La première étape a consisté au développement de la procédure de ‘protein design', en particulier, à la mise en place et à l'optimisation de la fonction d'énergie ainsi qu'à l'implémentation de l'algorithme d'optimisation. Nous avons montré que notre procédure est robuste puisqu'elle a fait ses preuves dans des applications très diverses telles que la prédiction de l'orientation des chaînes latérales, la prédiction des changements de stabilité ou d'affinité associés à des mutations ponctuelles, ou encore la production de séquences de type natif pour un jeu de protéines globulaires. Pour l'ensemble de ces applications, la qualité des résultats obtenus est comparable à celle observée chez d'autres groupes. Ensuite, nous avons appliqué notre procédure à des systèmes plus complexes tels que les systèmes protéine:ligand. Nous nous sommes intéressés à l'aspartyl-ARNt synthétase (AspRS) et l'asparaginyl-ARNt synthétase (AsnRS). Ces enzymes jouent un rôle crucial dans la traduction du code génétique. Les synthétases fixent leur acide aminé spécifique sur leur ARNt correspondant établissant ainsi l'intégrité du code génétique. Tout d'abord nous avons réalisé le ‘design' des sites actifs d'AspRS et d'AsnRS en présence de leur ligand natif et non natif afin d'évaluer les performances de notre procédure. La qualité des séquences prédites est comparable à celle observée pour les protéines globulaires entières. Par ailleurs, nous avons montré que notre procédure était sensible à la nature du ligand présent dans la poche. Enfin, nous avons réalisé le ‘design' d'un nombre limité de positions dans le site actif de l'AsnRS de façon à ce qu'elle lie préférentiellement l'aspartate au détriment de l'asparagine. Un jeu de mutants prometteurs fut retenu. Leur stabilité et affinité pour les ligands natifs et non natifs est actuellement analysé par des simulations de dynamique moléculaire.
• Detection of biological macromolecules on a biochip dedicated to UV specific absorption
  
  • Robin Kristelle
  • Reverchon Jean-Luc
  • Mugherli Laurent
  • Fromant Michel
  • Plateau Pierre
  • Benisty Henri
  Biosensors and Bioelectronics, Elsevier, 2008, 24 (6), pp.1585-1591. This work describes an ultraviolet biosensing technique based on specific molecular absorption detected with a previously developed spectrally selective aluminum gallium nitride (AlGaN) based detector. Light absorption signal of DNA and proteins, respectively at 260nm and 280nm, is used to image biochips. To allow detection of protein or DNA monolayers at the surface of a biochip, we develop contrast-enhancing multilayer substrates. We analyze them through models and experiments and validate the possibility of measuring absorptions of the order of 10(-3). These multilayer structures display a high reflectivity, and maximize the interaction of the electric field with the biological element at the chip surface. Optimization of the experimental absorption, which includes effects such as roughness of the biochip, spectral and angular resolution of the optics, illumination, etc., is carried out with an inorganic ultraviolet absorber (titanium dioxide) deposit. We obtained an induced absorption contrast enhanced by a factor of 4.0, conferring enough sensitivity to detect monolayers of DNA or proteins. Experimental results on an Escherichia coli histidine-tagged methionyl-tRNA synthetase protein before and after complexation with an anti-polyHis specific antibody validate our biosensing technique. This label-free optical method may be helpful in controlling biochip coatings, and subsequent biological coupling at the surface of a biochip. (10.1016/j.bios.2008.08.028)
  DOI : 10.1016/j.bios.2008.08.028
• Functional characterization of the Saccharomyces cerevisiae ABC-transporter Yor1p overexpressed in plasma membranes.
  
  • Grigoras Ioana
  • Lazard Myriam
  • Plateau Pierre
  • Blanquet Sylvain
  Biochimica et Biophysica Acta - Molecular Cell Research, Elsevier, 2008, 1778 (1), pp.68-78. Yor1p, a Saccharomyces cerevisiae plasma membrane ABC-transporter, is associated to oligomycin resistance and to rhodamine B transport. Here, by using the overexpressing strain Superyor [A. Decottignies, A.M. Grant, J.W. Nichols, H. de Wet, D.B. McIntosh, A. Goffeau, ATPase and multidrug transport activities of the overexpressed yeast ABC protein Yor1p, J. Biol. Chem. 273 (1998) 12612-12622], we show that Yor1p also confers resistance to rhodamine 6G and to doxorubicin. In addition, Yor1p protects cells, although weakly, against tetracycline, verapamil, eosin Y and ethidium bromide. The basal ATPase activity of the overexpressed form of Yor1p was studied in membrane preparations. This activity is quenched upon addition of micromolar amounts of vanadate. Vmax and Km values of approximately 0.8 s(-1) and 50+/-8 microM are measured. Mutations of essential residues in the nucleotide binding domain 2 reduces the activity to that measured with a Deltayor1 strain. ATP hydrolysis is strongly inhibited by the addition of potential substrates of the transporter. Covalent reaction of 8-azido-[alpha-(32)P]ATP with Yor1p is not sensitive to the presence of excess oligomycin. Thus, competition of the drug with ATP binding is unlikely. Finally, we inspect possible hypotheses accounting for substrate inhibition, rather than stimulation, of ATP hydrolysis by the membrane preparation. (10.1016/j.bbamem.2007.08.035)
  DOI : 10.1016/j.bbamem.2007.08.035
• Changes in the gene expression profile of Arabidopsis thaliana after infection with Tobacco etch virus.
  
  • Agudelo-Romero Patricia
  • Carbonell Pablo
  • de La Iglesia Francisca
  • Carrera Javier
  • Rodrigo Guillermo
  • Jaramillo Alfonso
  • Perez-Amador Miguel A.
  • Elena Santiago F.
  Virology Journal, BioMed Central, 2008, 5 (N/A), pp.92. BACKGROUND: Tobacco etch potyvirus (TEV) has been extensively used as model system for the study of positive-sense RNA virus infecting plants. TEV ability to infect Arabidopsis thaliana varies among ecotypes. In this study, changes in gene expression of A. thaliana ecotype Ler infected with TEV have been explored using long-oligonucleotide arrays. A. thaliana Ler is a susceptible host that allows systemic movement, although the viral load is low and syndrome induced ranges from asymptomatic to mild. Gene expression profiles were monitored in whole plants 21 days post-inoculation (dpi). Microarrays contained 26,173 protein-coding genes and 87 miRNAs. RESULTS: Expression analysis identified 1727 genes that displayed significant and consistent changes in expression levels either up or down, in infected plants. Identified TEV-responsive genes encode a diverse array of functional categories that include responses to biotic (such as the systemic acquired resistance pathway and hypersensitive responses) and abiotic stresses (droughtness, salinity, temperature, and wounding). The expression of many different transcription factors was also significantly affected, including members of the R2R3-MYB family and ABA-inducible TFs. In concordance with several other plant and animal viruses, the expression of heat-shock proteins (HSP) was also increased. Finally, we have associated functional GO categories with KEGG biochemical pathways, and found that many of the altered biological functions are controlled by changes in basal metabolism. CONCLUSION: TEV infection significantly impacts a wide array of cellular processes, in particular, stress-response pathways, including the systemic acquired resistance and hypersensitive responses. However, many of the observed alterations may represent a global response to viral infection rather than being specific of TEV. (10.1186/1743-422X-5-92)
  DOI : 10.1186/1743-422X-5-92
• Testing the Coulomb/Accessible Surface Area solvent model for protein stability, ligand binding, and protein design.
  
  • Schmidt Am Busch Marcel
  • Lopes Anne
  • Amara Najette
  • Bathelt C.
  • Simonson Thomas
  BMC Bioinformatics, BioMed Central, 2008, 9, pp.148. BACKGROUND: Protein structure prediction and computational protein design require efficient yet sufficiently accurate descriptions of aqueous solvent. We continue to evaluate the performance of the Coulomb/Accessible Surface Area (CASA) implicit solvent model, in combination with the Charmm19 molecular mechanics force field. We test a set of model parameters optimized earlier, and we also carry out a new optimization in this work, using as a target a set of experimental stability changes for single point mutations of various proteins and peptides. The optimization procedure is general, and could be used with other force fields. The computation of stability changes requires a model for the unfolded state of the protein. In our approach, this state is represented by tripeptide structures of the sequence Ala-X-Ala for each amino acid type X. We followed an iterative optimization scheme which, at each cycle, optimizes the solvation parameters and a set of tripeptide structures for the unfolded state. This protocol uses a set of 140 experimental stability mutations and a large set of tripeptide conformations to find the best tripeptide structures and solvation parameters. RESULTS: Using the optimized parameters, we obtain a mean unsigned error of 2.28 kcal/mol for the stability mutations. The performance of the CASA model is assessed by two further applications: (i) calculation of protein-ligand binding affinities and (ii) computational protein design. For these two applications, the previous parameters and the ones optimized here give a similar performance. For ligand binding, we obtain reasonable agreement with a set of 55 experimental mutation data, with a mean unsigned error of 1.76 kcal/mol with the new parameters and 1.47 kcal/mol with the earlier ones. We show that the optimized CASA model is not inferior to the Generalized Born/Surface Area (GB/SA) model for the prediction of these binding affinities. Likewise, the new parameters perform well for the design of 8 SH3 domain proteins where an average of 32.8% sequence identity relative to the native sequences was achieved. Further, it was shown that the computed sequences have the character of naturally-occuring homologues of the native sequences. CONCLUSION: Overall, the two CASA variants explored here perform very well for a wide variety of applications. Both variants provide an efficient solvent treatment for the computational engineering of ligands and proteins. (10.1186/1471-2105-9-148)
  DOI : 10.1186/1471-2105-9-148
• Homology modelling of protein-protein complexes: a simple method and its possibilities and limitations
  
  • Launay Guillaume
  • Simonson Thomas
  BMC Bioinformatics, BioMed Central, 2008, 9, pp.427.