Helical Inserts Research Articles

Properties of the C-terminal Domain of Enzyme I of the Escherichia coli Phosphotransferase System

The bacterial phosphoenolpyruvate (PEP):glycose phosphotransferase system (PTS) mediates uptake/phosphorylation of sugars. The transport of all PTS sugars requires Enzyme I (EI) and a phosphocarrier histidine protein of the PTS (HPr). The PTS is stringently regulated, and a potential mechanism is the monomer/dimer transition of EI, because only the dimer accepts the phosphoryl group from PEP. EI monomer consists of two major domains, at the N and C termini (EI-N and EI-C, respectively). EI-N accepts the phosphoryl group from phospho-HPr but not PEP. However, it is phosphorylated by PEP(Mg(2+)) when complemented with EI-C. Here we report that the phosphotransfer rate increases approximately 25-fold when HPr is added to a mixture of EI-N, EI-C, and PEP(Mg(2+)). A model to explain this effect is offered. Sedimentation equilibrium results show that the association constant for dimerization of EI-C monomers is 260-fold greater than the K(a) for native EI. The ligands have no detectable effect on the secondary structure of the dimer (far UV CD) but have profound effects on the tertiary structure as determined by near UV CD spectroscopy, thermal denaturation, sedimentation equilibrium and velocity, and intrinsic fluorescence of the 2 Trp residues. The binding of PEP requires Mg(2+). For example, there is no effect of PEP on the T(m), an increase of 7 degrees C in the presence of Mg(2+), and approximately 14 degrees C when both are present. Interestingly, the dissociation constants for each of the ligands from EI-C are approximately the same as the kinetic (K(m)) constants for the ligands in the complete PTS sugar phosphorylation assays.

Effects of axial baffles in non-circular multi-channel ceramic membranes using organic feed

The effect of axial baffles in non-circular channelled membranes on the critical flux has been investigated. Various geometric configurations of inserts, namely cylindrical rod, helical baffles and alternating direction helical baffles have been used. Higher critical fluxes were achieved with most of the inserts. Under the conditions of the present study i.e. at a fixed hydraulic dissipated power, the introduction of alternating direction helical baffles led to the greatest improvements (up to 37.7%). These improvements are ascribed to the turbulence generated and mixing that this encourages within the flow channel. Helical and cylindrical rod inserts produce comparable results which are unexpected and is attributed to the star-shaped flow channel which reduces the effectiveness of the rotational flow field that the helical baffles are thought to produce. In this study, helical baffles having two turns per 25 mm produce optimum flow conditions leading to the best performance in terms of the critical flux in the helical baffle series. An increase in the rod diameter seems to lead to a decrease in the flux enhancement. The results are explained in terms of the hydrodynamics that each axial baffle structure produces and their interaction with the star-shaped channel geometry within the filtration system.

Experimental studies on heat transfer and friction factor characteristics of laminar flow through a circular tube fitted with helical screw-tape inserts

Experimental investigation of heat transfer and friction factor characteristics of circular tube fitted with full-length helical screw element of different twist ratio, and increasing and decreasing order of twist ratio set have been studied with uniform heat flux. The experimental data obtained are compared with those obtained from plain tube published data. The heat transfer coefficient increases with twist ratio. There is no much change in the magnitude of heat transfer coefficient enhancement with increasing twist ratio and with decreasing twist ratio set, as the magnitude of swirl generated at the inlet or at the outlet in the order of increasing twist ratio or decreasing twist ratio, is same in both the cases. The empirical correlations developed in terms of twist ratio and Reynolds number, are fitting the experimental data within plus or minus 15% and 13% for Nusselt number and friction factor, respectively.

1H, 13C, and 15N Resonance Assignments for the Protein Coded by Gene Locus BB0938 of Bordetella bronchiseptica

The product of gene locus BB0938 from Bordetella bronchiseptica (Swiss-Prot ID: Q7WNU7-BORBR; NESG target ID: BoR11; Wunderlich et al., 2004; Pfam ID: PF03476) is a 128-residue protein of unknown function. This broadly conserved protein family is found in eubacteria and eukaryotes. Using triple resonance NMR techniques, we have determined 98% of backbone and 94% of side chain 1H, 13C, and 15N resonance assignments. The chemical shift and 3J(HN?Ha) scalar coupling data reveal a b topology with a seven-residue helical insert, ??????????. BMRB deposit with accession number 6693. Reference: Wunderlich et al. (2004) Proteins, 56, 181?187.

Roles for MreC and MreD proteins in helical growth of the cylindrical cell wall in Bacillus subtilis

Actin homologues of the MreB family have an important role in specifying the morphology of many non-spherical eubacteria. The mreC and mreD genes have been implicated in control of cell morphology but their precise functions are unknown. In Bacillus subtilis the MreB homologue Mbl directs helical insertion of new cell wall material in the cylindrical part of the rod-shaped cell. Depletion of either MreC or MreD abolishes the control of cell shape. In the presence of high concentrations of magnesium cells depleted of MreC or MreD can be propagated indefinitely, although they have a spheroidal shape. We show that growth of the spheroidal mutants is based on insertion of new wall material at cell division sites and that this localized growth is dependent on cell division. Under some conditions the MreC and MreD proteins localize in a helical configuration. This localization pattern resembles that of the helical cables of Mbl protein. These results suggest that MreC and MreD act in a morphogenic pathway that couples the helical cytosolic Mbl cables to the extracellular cell wall synthetic machinery, which is critical for cylindrical elongation of the rod-shaped cells.

Structure of the ectodomain of Drosophila peptidoglycan-recognition protein LCa suggests a molecular mechanism for pattern recognition

The peptidoglycan-recognition protein LCa (PGRP-LCa) is a transmembrane receptor required for activation of the Drosophila immune deficiency pathway by monomeric Gram-negative peptidoglycan. We have determined the crystal structure of the ectodomain of PGRP-LCa at 2.5-A resolution and found two unique helical insertions in the LCa ectodomain that disrupt an otherwise L-shaped peptidoglycan-docking groove present in all other known PGRP structures. The deficient binding of PGRP-LCa to monomeric peptidoglycan was confirmed by biochemical pull-down assays. Recognition of monomeric peptidoglycan involves both PGRP-LCa and -LCx. We showed that association of the LCa and LCx ectodomains in vitro depends on monomeric peptidoglycan. The presence of a defective peptidoglycan-docking groove, while preserving a unique role in mediating monomeric peptidoglycan induction of immune response, suggests that PGRP-LCa recognizes the exposed structural features of a monomeric muropeptide when the latter is bound to and presented by the ectodomain of PGRP-LCx. Such features include N-acetyl glucosamine and the anhydro bond in the glycan of the muropeptide, which have been demonstrated to be critical for immune stimulatory activity.

Structure of the SWI2/SNF2 chromatin-remodeling domain of eukaryotic Rad54

SWI2/SNF2 chromatin-remodeling proteins mediate the mobilization of nucleosomes and other DNA-associated proteins. SWI2/SNF2 proteins contain sequence motifs characteristic of SF2 helicases but do not have helicase activity. Instead, they couple ATP hydrolysis with the generation of superhelical torsion in DNA. The structure of the nucleosome-remodeling domain of zebrafish Rad54, a protein involved in Rad51-mediated homologous recombination, reveals that the core of the SWI2/SNF2 enzymes consist of two alpha/beta-lobes similar to SF2 helicases. The Rad54 helicase lobes contain insertions that form two helical domains, one within each lobe. These insertions contain SWI2/SNF2-specific sequence motifs likely to be central to SWI2/SNF2 function. A broad cleft formed by the two lobes and flanked by the helical insertions contains residues conserved in SWI2/SNF2 proteins and motifs implicated in DNA-binding by SF2 helicases. The Rad54 structure suggests that SWI2/SNF2 proteins use a mechanism analogous to helicases to translocate on dsDNA.

Flux Enhancement by Introducing Turbulence Effect for Microfiltration of Saccharomyces cerevisiae

Numerous studies have proven that the existence of turbulence effect could promote flux enhancement in the crossflow microfiltration channel. These turbulence effects can be generated by the introduction of turbulence promoters like helical baffles. Helical inserts reduce hold‐up in the feed channel; increase fluid velocity and wall shear rates; and produce secondary flows or instabilities. The aim of this work was to investigate the influence of turbulence effects in the feed channel on permeate flux during the microfiltration of Saccharomyces cerevisiae solutions. Tubular, single channel ceramic membranes with a nominal pore size of 0.2 µm were used. Variations of the helical baffle geometries, which are the number of turns per baffle length, were investigated. It was found that the insertion of helical baffles managed to increase the permeate flux. The optimum number of turns is four turns per 50 mm, which demonstrated the lowest cake resistance, R c , and highest permeation flux, J, for particular transmembrane pressure, ΔP. The increment of permeation flux reaches 88.2% while the cake resistance is reduced to 70.62% compared to the run without baffles.

Crystal Structure and Functional Implications of Pyrococcus furiosus Hef Helicase Domain Involved in Branched DNA Processing

DNA and RNA frequently form various branched intermediates that are important for the transmission of genetic information. Helicases play pivotal roles in the processing of these transient intermediates during nucleic acid metabolism. The archaeal Hef helicase/ nuclease is a representative protein that processes flap- or fork-DNA structures, and, intriguingly, its C-terminal half belongs to the XPF/Mus81 nuclease family. Here, we report the crystal structure of the helicase domain of the Hef protein from Pyrococcus furiosus. The structure reveals a novel helical insertion between the two conserved helicase core domains. This positively charged extra region, structurally similar to the "thumb" domain of DNA polymerase, plays critical roles in fork recognition. The Hef helicase/nuclease exhibits sequence similarity to the Mph1 helicase from Saccharomyces cerevisiae; XPF/Rad1, involved in DNA repair; and a putative Hef homolog identified in mammals. Hence, our findings provide a structural basis for the functional mechanisms of this helicase/nuclease family.

Crystal Structure of the Phosphoenolpyruvate-binding Enzyme I-Domain from the Thermoanaerobacter tengcongensis PEP: Sugar Phosphotransferase System (PTS)

Enzyme I (EI), the first component of the phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS), consists of an N-terminal protein-binding domain (EIN) and a C-terminal PEP-binding domain (EIC). EI transfers phosphate from PEP by double displacement via a histidine residue on EIN to the general phosphoryl carrier protein HPr. Here, we report the 1.82A crystal structure of the homodimeric EIC domain from Thermoanaerobacter tengcongensis, a saccharolytic eubacterium that grows optimally at 75 degrees C. EIC folds into a (betaalpha)(8) barrel with three large helical insertions between beta2/alpha2, beta3/alpha3 and beta6/alpha6. The large amphipathic dimer interface buries 3750A(2) of accessible surface area per monomer. A comparison with pyruvate phosphate dikinase (PPDK) reveals that the active-site residues in the empty PEP-binding site of EIC and in the liganded PEP-binding site of PPDK have almost identical conformations, pointing to a rigid structure of the active site. In silico models of EIC in complex with the Z and E-isomers of chloro-PEP provide a rational explanation for their difference as substrates and inhibitors of EI. The EIC domain exhibits 54% amino acid sequence identity with Escherichia coli and 60% with Bacillus subtilis EIC, has the same amino acid composition but contains additional salt-bridges and a more complex salt-bridge network than the homology model of E.coli EIC. The easy crystallization of EIC suggests that T.tengcongensis can serve as source for stable homologs of mesophilic proteins that are too labile for crystallization.

Enhancement of heat transfer in a tube with regularly-spaced helical tape swirl generators

Influence of helical tapes inserted in a tube on heat transfer enhancement is studied experimentally. A helical tape is inserted in the tube with a view to generating swirl flow that helps to increase the heat transfer rate of the tube. The flow rate of the tube is considered in a range of Reynolds number between 2300 and 8800. The swirling flow devices consisting of: (1) the full-length helical tape with or without a centered-rod, and (2) the regularly-spaced helical tape, are inserted in the inner tube of a concentric tube heat exchanger. Hot air is passed through the inner tube whereas cold water is flowed in the annulus. The experimental data obtained are compared with those obtained from plain tubes of published data. Experimental results confirmed that the use of helical tapes leads to a higher heat transfer rate over the plain tube. The full-length helical tape with rod provides the highest heat transfer rate about 10% better than that without rod but it increased the pressure drop. To overcome this, different free-spacing ratio ( s = L s/ L h) of 0.5, 1.0, 1.5, and 2.0 were examined. It was found that the space ratio value should be about unity for Re < 4000. The regularly-spaced helical tape inserts at s = 0.5 yields the highest Nusselt number which is about 50% above the plain tube.

Baffled microfiltration membrane and its fouling control for feed water of desalination

Pretreatment process is a significant factor in the success of desalination process. It plays a key role in enhancing the lifetime of RO membranes in desalination process. Microfiltration process is emerging as an alternative solution for desalination pretreatment process. The baffled microfiltration membrane has its attractive features in fouling control and flux improvement for the feed water for desalination. Turbulence promoters such as helical baffles have distinct possibilities to enhance permeate flux during crossflow microfiltration. Helical inserts reduce hold-up in the feed channel; increase fluid velocity and wall shear rates and produce secondary flows or instabilities. The overall objective of this work was to investigate the influence of helical baffles on permeate flux during the microfiltration process. TiO 2 dispersion was used to represent the microfiltration particle. Tubular, single channel ceramic membranes with nominal pore size of 0.2 μm were used. Variations of the helical baffle geometries, which are the number of turns per baffle length, were investigated. It was found that the insertion of helical baffles had increased the permeate flux. In some cases, the increase was more than 100%. The effect of a number of turns per baffle length showed that the permeate flux increased with the number of turns but decreased when the number of turns was more than 4 turns per 50 mm baffle length. Thus, the optimum number of turns was 4 turns per 50 mm.

A morphometric study of the external ear: age- and sex-related differences.

The human ear is a defining feature of the face. Its subtle structures convey signs of age and sex that are unmistakable yet not easily defined. With analysis of normative cross-sectional data, this study explored anatomic and aesthetic differences in the ear between men and women, as well as changes in ear morphology with age. A total of 123 volunteers were randomly selected for this study. The cohort consisted of 89 women ages 19 to 65 years (median age, 42 years) and 34 men ages 18 to 61 years (median age, 35 years). The average total ear height across the entire cohort for both left and right ears was 6.30 cm, average lobular height was 1.88 cm, and average lobular width was 1.96 cm. As expected based on head size, significant sex-related differences were noted in the distance from the lateral palpebral commissure to both the helical root and insertion of the lobule. Measured distances in both vectors were approximately 4.6 percent longer in men than in women. Similarly, the height of the pinna was significantly larger in men than in women by approximately 6.5 percent. The average height and width of the lobule, however, were nearly identical in men and women. Analysis of age-related data showed a significant difference in the total ear height between the subpopulations; however, this difference was not significant after the lobular height was subtracted from total ear height, suggesting that the lobule was the only ear structure that changed significantly with age. In addition, lobular width decreased significantly with age. This study establishes normative data for ear morphology and clearly demonstrates the changes in earlobe morphology that occur with advancing age.

Insights into antifolate resistance from malarial DHFR-TS structures.

Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) is an important target of antimalarial drugs. The efficacy of this class of DHFR-inhibitor drugs is now compromised because of mutations that prevent drug binding yet retain enzyme activity. The crystal structures of PfDHFR-TS from the wild type (TM4/8.2) and the quadruple drug-resistant mutant (V1/S) strains, in complex with a potent inhibitor WR99210, as well as the resistant double mutant (K1 CB1) with the antimalarial pyrimethamine, reveal features for overcoming resistance. In contrast to pyrimethamine, the flexible side chain of WR99210 can adopt a conformation that fits well in the active site, thereby contributing to binding. The single-chain bifunctional PfDHFR-TS has a helical insert between the DHFR and TS domains that is involved in dimerization and domain organization. Moreover, positively charged grooves on the surface of the dimer suggest a function in channeling of substrate from TS to DHFR active sites. These features provide possible approaches for the design of new drugs to overcome antifolate resistance.

Mutational analysis of tetracycline resistance protein transmembrane segment insertion

The tetracycline resistance proteins (TetA) of gram-negative bacteria are secondary active transport proteins that contain buried charged amino acids that are important for tetracycline transport. Earlier studies have shown that insertion of TetA proteins into the cytoplasmic membrane is mediated by helical hairpin pairs of transmembrane (TM) segments. However, whether helical hairpins direct spontaneous insertion of TetA or are required instead for its interaction with the cellular secretion (Sec) machinery is unknown. To gain insight into how TetA proteins are inserted into the membrane, we have investigated how tolerant the class C TetA protein encoded by plasmid pBR322 is to placement of charged residues in TM segments. The results show that the great majority of charge substitutions do not interfere with insertion even when placed at locations that cannot be shielded internally within helical hairpins. The only mutations that frequently block insertion are proline substitutions, which may interfere with helical hairpin folding. The ability of TetA to broadly tolerate charge substitutions indicates that the Sec machinery assists in its insertion into the membrane. The results also demonstrate that it is feasible to engineer charged residues into the interior of TetA proteins for the purpose of structure–function analysis.

Flux enhancement in microfiltration by corkscrew vortices formed in helical flow passages

The performance of helical inserts in existing tubular filters has been investigated by Bellhouse et al. [12]. The insert and filter combination was found to deliver high filtration fluxes at low cross-flow rates over a wide range of applications, covering reverse osmosis to microfiltration. In addition the flow shear stresses were viscous, and fluids containing delicate organisms, such as marine algae, were concentrated without damage. The increased fluxes were attributed to the generation of corkscrew vortices, formed by the interaction of Dean vortices and an axial flow component. If this explanation is correct, it should be possible to generate similar corkscrew vortices in internal helical passages, which surround a central lumen. By coating the internal surface of the helical passages with a suitable membrane, a one-piece filter, capable of emulating the performance of the insert and tubular filter, could be constructed. Ruiz et al. [16] had demonstrated their ability to produce ceramic filters with internal helical grooves, and the Laboratoire des Materiaux et Procedes Membranaires at Montpellier University produced a number of prototype filters to a design derived from the geometries used in [12]. These filters were tested using high concentrations of bakers’ yeast. Similar fluxes to those obtained with the inserts and tubular filters were achieved, over the same range of flow rates. Whilst the mechanism of concentration polarisation reduction by corkscrew vortices works over the whole range of filtration applications, the unique combination of viscous flow, high fluxes, low cross-flow rates and high feed concentrations is particularly suited to microfiltration in bio-processing applications.

Defining cosQ, the site required for termination of bacteriophage lambda DNA packaging.

Bacteriophage lambda is a double-stranded DNA virus that processes concatemeric DNA into virion chromosomes by cutting at specific recognition sites termed cos. A cos is composed of three subsites: cosN, the nicking site; cosB, required for packaging initiation; and cosQ, required for termination of chromosome packaging. During packaging termination, nicking of the bottom strand of cosN depends on cosQ, suggesting that cosQ is needed to deliver terminase to the bottom strand of cosN to carry out nicking. In the present work, saturation mutagenesis showed that a 7-bp segment comprises cosQ. A proposal that cosQ function requires an optimal sequence match between cosQ and cosNR, the right cosN half-site, was tested by constructing double cosQ mutants; the behavior of the double mutants was inconsistent with the proposal. Substitutions in the 17-bp region between cosQ and cosN resulted in no major defects in chromosome packaging. Insertional mutagenesis indicated that proper spacing between cosQ and cosN is required. The lethality of integral helical insertions eliminated a model in which DNA looping enables cosQ to deliver a gpA protomer for nicking at cosN. The 7 bp of cosQ coincide exactly with the recognition sequence for the Escherichia coli restriction endonuclease, EcoO109I.

The performance of helical screw-thread inserts in tubular membranes

Abstract A detailed study of the performance of helical screw-thread inserts in tubular membranes has been carried out at the University of Oxford. The design combines a predominantly helical flow, in which Dean vortices are generated, with an approximately axial flow, which modifies the Dean vortices into a continuous ‘corkscrew vortex’. This promotes good mixing of the feed fluid and minimises concentration polarisation effects. Computational fluid dynamics (CFD) modelling of the flow has been carried out. Comparisons of the performance of plain membranes with membranes fitted with various inserts are reported. The behaviour of the screw thread inserts was modelled using the commercial CFD code ‘Fluent’. Good agreement was found between the CFD predictions and experimental measurements. Laminar flow conditions appear to be maintained at high cross-flow rates, suggesting that the helical insert geometry could improve the filtration of shear-sensitive fluids such as blood. Measurements using bovine blood and suspensions of marine algae supported this conclusion. Testing of tubular membranes fitted with one-start and three-start helical inserts has been completed. The inserts were tested under microfiltration conditions using yeast solutions, under ultrafiltration conditions using reconstituted powdered milk solutions, and under nano filtration conditions using synthetic dyes. Two pumped loops were built for these experiments. The helical inserts produced much higher fluxes at low cross-flow rates than membranes without inserts (up to a factor of 6 higher), and also performed better than a plain cylindrical insert which had a similar pressure drop versus flow characteristic. Thus helical inserts improve the filtration of shear-sensitive fluids at low cross-flows; they also produce high fluxes with low cross-flow rates, which is beneficial if high value products are being separated. By suitably modifying the geometry of the insert, lower fluxes and pressure drops can be obtained: this offers the opportunity of tailoring the insert performance to the requirements of a particular process.

Residence time distribution studies in a simulated tubular affinity membrane separator

One of the key factors in designing an affinity membrane contactor is the flow pattern in the device. This paper presents a study carried out on the residence time distribution (RTD) of a tracer in a cross-flow tubular affinity module. For simplicity, no membrane was incorporated in the system. A tubular conduit with a total length of 0.7 m was designed and to improve the RTD of the tracer, various screw-threaded inserts were tested. The experimental tests for the RTDs in a channel with no inserts, a channel with a plain rod and a channel with helical inserts were performed and compared with the corresponding theoretical models. Two different methods of analyzing the data were adopted. The first method was based on the evaluation of the variance of the E-curves, and the second was based on the use of Fourier transform analysis. The experimental data showed that appropriate combination of pulsation frequency, piston stroke length, and geometry resulted in an optimum value for the Peclet number. This provides an explanation of why the capturing efficiency of the target species in membrane affinity separations is enhanced significantly by oscillatory flow.

Crystal structure of the hydrogenase maturating endopeptidase HYBD from Escherichia coli

The maturation of [NiFe] hydrogenases includes formation of the nickel metallocenter, proteolytic processing of the metal center carrying large subunit, and its assembling with other hydrogenase subunits. The hydrogenase maturating enzyme HYBD from Escherichia coli, a protease of molecular mass 17.5 kDa, specifically cleaves off a 15 amino acid peptide from the C terminus of the precursor of the large subunit of hydrogenase 2 in a nickel-dependent manner. Here we report the crystal structure of HYBD at 2.2 Å resolution. It consists of a twisted five-stranded β-sheet surrounded by four and three helices, respectively, on each side. A cadmium ion from the crystallization buffer binds to the proposed nickel-binding site and is penta-coordinated by Glu16, Asp62, His93, and a water molecule in a pseudo-tetragonal arrangement. HYBD is topologically related to members of the metzincins superfamily of zinc endoproteinases, sharing the central β-sheet and three helices. In contrast to the metzincins, the metal-binding site of HYBD is localized at the C-terminal end of the β-sheet. Three helical insertions unique to HYBD pack against one side of the sheet, build up the active site cleft, and provide His93 as ligand to the metal. From this structure, we derive molecular clues into how the protease HYBD is involved in the hydrogenase maturation process.