Dissecting the maturation steps of the lasso peptide microcin J25 in vitro

Microcin J25 is the archetype of a growing class of bacterial ribosomal peptides possessing a knotted topology (lasso peptides). It consists of an eight-residue macrolactam ring through which the C-terminal tail is threaded. It is biosynthesized as a precursor that is processed by two maturation enzymes (McjB/McjC). Insights into the mechanism of microcin J25 biosynthesis have been provided previously by mutagenesis of the precursor peptide in vivo. In this study we have demonstrated distinct functions of McjB and McjC in vitro for the first time, based on the detection of reaction intermediates. McjB was characterized as a new ATP-dependent cysteine protease, whereas McjC was confirmed to be a lactam synthetase. The two enzymes were functionally interdependent, likely forming a structural complex. Their substrate preference was directly investigated with the aid of mutated precursor peptides. Depending on the substitutions, microcin J25 variants with either a lasso or branched-cyclic topology could be generated in vitro.     

Construction of a single polypeptide that matures and exports the lasso peptide microcin J25

Roped in: The lasso peptide microcin J25 (MccJ25) is matured by two enzymes and is exported by a putative ABC transporter. We probed the function of the maturation enzymes using mutagenesis. We demonstrate that fusions of the enzymes with intervening linkers can produce MccJ25. Even a 151 kDa tripartite fusion between the ABC transporter and the two enzymes is capable of producing and exporting MccJ25.     

Sequence determinants governing the topology and biological activity of a lasso peptide, microcin J25

Microcin J25 is a potent antibacterial peptide produced by Escherichia coli AY25. It displays a lasso structure, which consists of a knot involving an N-terminal macrolactam ring through which the C-terminal tail is threaded and sterically trapped. In this study, we rationally designed and performed site-specific mutations in order to pinpoint the sequence determinants of the lasso topology. Structures of the resulting variants were analysed by a combination of methods (mass spectrometry, NMR spectroscopy, enzymatic digestion), and correlated to the antibacterial activity. The selected mutations resulted in the production of branched-cyclic or lasso variants. The C-terminal residues below the ring (Tyr20, Gly21) and the size of the macrolactam ring were revealed to be critical for both the lasso scaffold and bioactivity, while shortening the loop region (Tyr9-Ser18) or extending the C-terminal tail below the ring did not alter the lasso structure, but differentially affected the antibacterial activity. These results provide new insights for the bioengineering of antibacterial agents using a lasso peptide as template.     

Computational design of reduced metabolic networks

Cellular functions are based on thousands of chemical reactions and transport processes, most of them being catalysed and regulated by specific proteins. Systematic gene knockouts have provided evidence that this complex reaction network possesses considerable redundancy, that is, alternative routes exist along which signals and metabolic fluxes may be directed to accomplish an identical output behaviour. This property is of particular importance in cases where parts of the reaction network are transiently or permanently impaired, for example, due to an infection or genetic alterations. Here we present a computational concept to determine enzyme-reduced metabolic networks that are still sufficient to accomplish a given set of cellular functions. Our approach consists of defining an objective function that expresses the compromise that has to be made between successive reduction of the network by omission of enzymes and its decreasing thermodynamic and kinetic feasibility. Optimisation of this objective function results in a linear mixed-integer program. With increasing weight given to the reduction of the number of enzymes, the total flux in the network increases and some of the reactions have to proceed in thermodynamically unfavourable directions. The approach was applied to two metabolic schemes: the energy and redox metabolism of red blood cells and the carbon metabolism of Methylobacterium extorquens. For these two example networks, we determined various variants of reduced networks differing in the number and types of disabled enzymes and disconnected reactions. Using a comprehensive kinetic model of the erythrocyte metabolism, we assess the kinetic feasibility of enzyme-reduced subnetworks. The number of enzymes predicted to be indispensable amounts to 14 (out of 28) for the erythrocyte scheme and 13 (out of 77) for the bacterium scheme, the largest group of enzymes predicted to be simultaneously dispensable amounts to 3 and 37 for these two systems. Our approach might contribute to identifying potential target enzymes for rational drug design, to rationalising gene-expression profiles of metabolic enzymes and to designing synthetic networks with highly specialised metabolic functions.     

J型烟囱的设计介绍

介绍为６．３ｔ／ｈ燃油锅炉配套的采用直接排出方式的Ｊ型烟囱．阐述了此烟囱的结构和高度设计的系列计算．     

Computational design of an endo-1,4-beta-xylanase ligand binding site

The field of computational protein design has experienced important recent success. However, the de novo computational design of high-affinity protein-ligand interfaces is still largely an open challenge. Using the Rosetta program, we attempted the in silico design of a high-affinity protein interface to a small peptide ligand. We chose the thermophilic endo-1,4-β-xylanase from Nonomuraea flexuosa as the protein scaffold on which to perform our designs. Over the course of the study, 12 proteins derived from this scaffold were produced and assayed for binding to the target ligand. Unfortunately, none of the designed proteins displayed evidence of high-affinity binding. Structural characterization of four designed proteins revealed that although the predicted structure of the protein model was highly accurate, this structural accuracy did not translate into accurate prediction of binding affinity. Crystallographic analyses indicate that the lack of binding affinity is possibly due to unaccounted for protein dynamics in the 'thumb' region of our design scaffold intrinsic to the family 11 β-xylanase fold. Further computational analysis revealed two specific, single amino acid substitutions responsible for an observed change in backbone conformation, and decreased dynamic stability of the catalytic cleft. These findings offer new insight into the dynamic and structural determinants of the β-xylanase proteins.     

Computational design of molecular properties: spotlight on accuracy and tuning

The Laboratory for Computational Molecular Design at ISIC devises original and accurate methodologies to establish, in silico, key structure-property relationships of large chemical systems with particular emphasis on those associated with pi-conjugated framework. Herein, we discuss two specific focuses of our activities: i) the development of accurate formalisms based on Kohn-Sham density functional theory to achieve quantitative results for the energies and geometries of extended systems featuring weak interactions and ii) the introduction of schemes to probe and tune the effect of intra- and intermolecular charge transfer on molecular properties. The proposed methodologies are ideally designed to tackle and resolve some of today's relevant aspects associated with the properties of pi-conjugated molecules, such as identifying relationships resulting in high stacking capacities, proposing more stable alternative topologies to large acenes, and analyzing the course of reaction involving assemblies of pi-conjugated molecules.     

Computational fluid dynamics‐based design of finned steam cracking reactors

The use of one‐dimensional reactor models to simulate industrial steam cracking reactors has been one of the main limiting factors for the development of new reactor designs and the evaluation of existing three‐dimensional (3‐D) reactor configurations. Therefore, a 3‐D computational fluid dynamics approach is proposed in which the detailed free‐radical chemistry is for the first time accounted for. As a demonstration case, the application of longitudinally and helicoidally finned tubes as steam cracking reactors was investigated under industrially relevant conditions. After experimental validation of the modeling approach, a comprehensive parametric study allowed to identify optimal values of the fin parameters, that is, fin height, number of fins, and helix angle to maximize heat transfer. Reactive simulations of an industrial Millisecond propane cracker were performed for four distinct finned reactors using a reaction network of 26 species and 203 elementary reactions. The start‐of‐run tube metal skin temperatures could be reduced by up to 50 K compared to conventionally applied tubular reactors when applying optimal fin parameters. Implementation of a validated coking model for light feedstocks shows that coking rates are reduced up to 50%. However, the increased friction and inner surface area lead to pressure drops higher by a factor from 1.22 to 1.66 causing minor but significant shifts in light olefin selectivity. For the optimized helicoidally finned reactor the ethene selectivity dropped, whereas propene and 1,3‐butadiene selectivity increased with a similar amount. The presented methodology can be applied in a straightforward way to other 3‐D reactor designs and can be extended to more complex feedstocks such as naphtha. © 2013 American Institute of Chemical Engineers AIChE J 60: 794–808, 2014     

Computational design of molecularly imprinted polymer for direct detection of melamine in milk

A novel protocol for use of molecularly imprinted polymer (MIP) in analysis of melamine is presented. Design of polymer for melamine has been achieved using a combination of computational techniques and laboratory trials, the former greatly reducing the duration of the latter. The compatibility and concerted effect of monomers and solvents were also investigated and discussed. Two novel open-source tools were presented which are the online polymer calculator from mipdatabase.com and the application of the Gromacs modelling suite to determine the ideal stoichiometric ratio between template and functional monomer. The MIP binding was characterised for several structural analogues at 1–100 μM concentrations. The use of divinylbenzene (DVB) as cross-linking polymer and itaconic acid as functional monomer allowed synthesis of MIP with imprint factor (IF) of 2.25 for melamine. This polymer was used in high-performance liquid chromatography (HPLC) for the rapid detection of melamine in spiked milk samples with an experimental run taking 7–8 min. This approach demonstrated the power of virtual tools in accelerated design of MIPs for practical applications.     

Computational design of thermoset nanocomposite coatings: Methodological study on coating development and testing

Thermoset nanocomposites (TSNCs) may offer significantly improved performance over conventional thermoset materials, and thus are attractive for wide industrial applications, especially in the coating industry. Design of TSNCs via experiment, however, faces various technical challenges due to design complexity. Computational design can provide deep insights and identify superior design solutions through exploring opportunities in a usually huge design space. This paper introduces a generic computational methodology for the design, characterization, and testing of TSNC-based coatings. A distinct feature of the methodology is its capability of generating quantitative correlations among material formulation, processing condition, coating microstructure and property, coating performance, and processing efficiency. The correlations can enable a comprehensive analysis for optimal TSNC coating design. Case studies will demonstrate the methodological efficacy and attractiveness.     

Initial Molecular Recognition Steps of McjA Precursor during Microcin J25 Lasso Peptide Maturation

Microcin J25 (MccJ25) has emerged as an excellent model to understand the maturation of ribosomal precursor peptides into the entangled lasso fold. MccJ25 biosynthesis relies on the post‐translational modification of the precursor McjA by the ATP‐dependent protease McjB and the lactam synthetase McjC. Here, using NMR spectroscopy, we showed that McjA is an intrinsically disordered protein without detectable conformational preference, which emphasizes the active role of the maturation machinery on the three‐dimensional folding of MccJ25. We further showed that the N‐terminal region of the leader peptide is involved in interaction with both maturation enzymes and identified a predominant interaction of V43–S55 in the core McjA sequence with McjC. Moreover, we demonstrated that residues K23–Q34 in the N‐terminal McjA leader peptide tend to adopt a helical conformation in the presence of membrane mimics, implying a role in directing McjA to the membrane in the vicinity of the lasso synthetase/export machinery. These data provide valuable insights into the initial molecular recognition steps in the MccJ25 maturation process.     

A method for computational combinatorial peptide design of inhibitors of Ras protein

A computational combinatorial approach is proposed for the designof a peptide inhibitor of Ras protein. The procedure involvesthree steps. First, a `Multiple Copy Simultaneous Search' identifiesthe location of specific functional groups on the Ras surface.This search method allowed us to identify an important bindingsurface consisting of two ß strands (residues 5–8and 52–56), in addition to the well known Ras effectorloop and switch II region. The two ß strands had not previouslybeen reported to be involved in Ras–Raf interaction. Second,after constructing the peptide inhibitor chain based on thelocation of N-methylacetamide (NMA) minima, functional groupsare selected and connected to the main chain C atom. This stepgenerates a number of possible peptides with different sequenceson the Ras surface. Third, potential inhibitors are designedbased on a sequence alignment of the peptides generated in thesecond step. This computational approach reproduces the conservedpattern of hydrophobic, hydrophilic and charged amino acidsidentified from the Ras effectors. The advantages and limitationsof this approach are discussed.     

Computational studies of the gramicidin channel

Ion channels are highly specific membrane-spanning protein structures which serve to facilitate the passage of selected ions across the lipid barrier. In the past decade, molecular dynamics simulations based on atomic models and realistic microscopic interactions with explicit solvent and membrane lipids have been used to gain insight into the function of these complex systems. These calculations have considerably expanded our view of ion permeation at the microscopic level. This Account will mainly focus on computational studies of the gramicidin A channel, one of the simplest and best characterized molecular pore.     

Conceptual design of distillation processes for mixtures with distillation boundaries: I. Computational assessment of split feasibility

Geometric design methods for the conceptual design of azeotropic distillation processes are fast and efficient tools for the economic screening of different process alternatives. This two‐part series presents a fully automated conceptual design method for finding an optimal recycle policy for the separation of mixtures with distillation boundaries. It does not require visualization and graphical inspection of residue curve or pinch maps and is, hence, not limited to ternary mixtures. The first part introduces a fully computational geometric split feasibility test based on bifurcation analysis. This bifurcation‐based feasibility test can be used as a valuable stand‐alone tool for the assessment of different separation options. It is also one of the core elements of the recycle optimization discussed in the second part of this series. © 2010 American Institute of Chemical Engineers AIChE J, 2011     

The use of multivariate analysis for the design of selective isolation conditions for mutants of Streptomyces cattleya with improved antibiotic titre

Twenty-three tests which did not include antibiotic production were carried out on ultraviolet-induced mutants of Streptomyces cattleya NRRL 8057. Principal component analysis of the results indicated the taxometric relationships between members of a population consisting of 77 isolates. Highlitre antibiotic producers were then marked on the population distribution diagram and clusters containing high-titre antibiotic producers noted. Discriminant analysis for the high-titre clusters provided a discriminant function which was derived from data from seven of the tests. These were used to formulate a positive selection procedure which was successful in selecting for a population of mutants which contained a significantly higher number of high-titre isolates than a control population.     

Computational investigation of the mechanical properties of nanomaterials

The mechanical responses of carbon nanotubes are examined using classical molecular dynamics simulations. Several different types of nanotubes are considered, including pristine single-walled tubes that are empty, filled with fullerenes to form peapods, filled with other nanotubes to form multi-walled tubes, or chemically functionalized. In addition, the responses of single-walled nanotubes with wall vacancies are considered. The results show how the bending force of filled nanotubes increases relative to the bending force of empty nanotubes and indicates how these increases come about. In addition, the simulations reveal the way in which the magnitude of these increases depend on the type of filling material and, in the case of multi-walled tubes, the number of inner tubes. These simulations further illustrate the way in which the inner nanotubes support higher external loads than the fullerenes in cases when the outer nanotubes are identical. The results also indicate that both the bending and buckling forces depend on temperature and the reasons for this dependence are discussed. Lastly, the simulations demonstrate the way in which the introduction of vacancy defects and covalently bound functional groups to the nanotube walls degrades the nanotubes' mechanical properties.     

TATB晶体形貌的计算模拟

利用Materials Studio中Morphology模块所含的BFDH、Growth和Equilibrium morphology的3种方法计算了TATB的晶体形态和结晶习性,得到特定晶面的面积、附着能、表面能及晶面相对生长速率等参数,分析了重要晶面的结构、晶体的生长习性和晶面结构与晶形控制剂的关系。结果表明,选择官能团中含有活泼H原子和O原子的表面活性剂,可更有效地控制TATB晶体的形态。