Wear lip formation during dry sliding

A pin-on-disc test configuration has been used to examine the formation of the strain-hardened projection, or wear lips, especially at the trailing edge of the pin during dry sliding of aluminium alloys against steel discs. The mechanism of formation of such wear lips is studied with the aid of optical and electron microscopes. The plastic deformation of the pin, growth and eventual removal of the wear lip as wear debris are elucidated. The size and shape of the wear lips in pins of different shapes, i.e. square, rectangular, triangular and circular cross-sections, are described.

A phase field study of morphological instabilities in multilayer thin films

We studied the microstructural evolution of multiple layers of elastically stiff films embedded in an elastically soft matrix using a phase field model. The coherent and planar film/matrix interfaces are rendered unstable by the elastic stresses due to a lattice parameter mismatch between the film and matrix phases, resulting in the break-up of the films into particles. With an increasing volume fraction of the stiff phase, the elastic interactions between neighbouring layers lead to: (i) interlayer correlations from an early stage; (ii) a longer wavelength for the maximally growing wave; and therefore (iii) a delayed break-LIP. Further, they promote a crossover in the mode of instability from a predominantly anti-symmetric (in phase) one to a symmetric (out of phase) one. We have computed a stability diagram for the most probable mode of break-up in terms of elastic modulus Mismatch and Volume fraction. We rationalize our results in terms of the initial driving force for destabilization, and corroborate our conclusions using simulations in elastically anisotropic systems.

Chemical probes into the active centre of a heme thiolate monoxygenase

Linalool-8-monoxygenase, a typical bacterial P-450 heme thiolase, shows a high degree of substrate specificity towards linalool. The active site of the pure enzyme has been probed with a large number of substrate analogues with systematic alterations or conformational variations in the linalool molecule. The comparison of three parameters, the mo→mos conversion of the enzyme as a result of substrate binding monitored at 392 nm, theK D of the analogues giving information about energies of association and the relative turnover as substrate have given information about the space-filling characteristics of the substrates in the enzyme cleft, the number of contacts the molecules make with the respective domains of the enzyme and the distance of the site undergoing hydroxylation from the oxygen site, respectively. The data permit the conclusion that linalool makes contact with the enzyme by hydrogen bonding with the hydroxyl group as well through hydrophobic association with all the eight carbons carrying hydrogen in the molecules.

Additional binding sites in lysozyme. X-ray analysis of lysozyme complexes with bromophenol red and bromophenol blue

The binding sites in hen egg-white lysozyme for neutral bromophenol red (BPR) and ionized bromophenol blue (BPB) have been characterized at 2 Å resolution. In either case, the dye-bound enzyme is active against the polysaccharide, but not against the cell wall. Both binding sites are outside, but close to, the hexasaccharide binding cleft in the enzyme. The binding site of BPR made up of Arg5, Lys33, Phe34, Asn37, Phe38, Ala122, Trp123 and possibly Arg125, is dose to subsite F while that of BPB made up of Tyr20, Arg21, Asn93, Lys96, Lys97 and Ser100, is close to subsites A and B. The binding sites of the neutral dye and the ionized dye are thus spatially far apart. The peptide component of the bacterial cell wall probably interacts with these cells during enzyme action. Such interactions are perhaps necessary for appropriately positioning the enzyme molecule on the bacterial cell wall.

Functional characterization of the precursor and spliced forms of RecA protein of Mycobacterium tuberculosis

The recA locus of pathogenic mycobacteria differs from that of nonpathogenic species because it contains large intervening sequences nested in the RecA homology region that are excised by an unusual protein-splicing reaction. In vivo assays indicated that Mycobacterium tuberculosis recA partially complemented Escherichia coli recA mutants for recombination and mutagenesis. Further, splicing of the 85 kDa precursor to 38 kDa MtRecA protein was necessary for the display of its activity, in vivo. To gain insights into the molecular basis for partial and lack of complementation by MtRecA and 85 kDa proteins, respectively, we purified both of them to homogeneity. MtRecA protein, but not the 85 kDa form, bound stoichiometrically to single-stranded DNA in the presence of ATP. MtRecA protein was cross-linked to 8-azidoadenosine 5'-triphosphate with reduced efficiency, and kinetic analysis of ATPase activity suggested that it is due to decreased affinity for ATP. In contrast, the 85 kDa form was unable to bind ATP, in the presence or absence of ssDNA and, consequently, was entirely devoid of ATPase activity. Molecular modeling studies suggested that the decreased affinity of MtRecA protein for ATP and the reduced efficiency of its hydrolysis might be due to the widening of the cleft which alters the hydrogen bonds and the contact area between the enzyme and the substrate and changes in the disposition of the amino acid residues around the magnesium ion and the gamma-phosphate. The formation of joint molecules promoted by MtRecA protein was stimulated by SSB when the former was added first. The probability of an association between the lack and partial levels of biological activity of RecA protein(s) to that of illegitimate recombination in pathogenic mycobacteria is considered.

Structural plasticity and enzyme action: Crystal structures of Mycobacterium tuberculosis peptidyl-tRNA hydrolase

Peptidyl-tRNA hydrolase cleaves the ester bond between tRNA and the attached peptide in peptidyl-tRNA in order to avoid the toxicity resulting from its accumulation and to free the tRNA available for further rounds in protein synthesis. The structure of the enzyme from Mycobacteritan tuberculosis has been determined in three crystal forms. This structure and the structure of the enzyme frorn Escherichia coli in its crystal differ substantially on account of the binding of the C terminus of the E. coli enzyme to the peptide-binding site of a neighboring molecule in the crystal. A detailed examination of this difference led to an elucidation of the plasticity of the binding site of the enzyme. The peptide-binding site of the enzyme is a cleft between the body, of the molecule and a polypepticle Y stretch involving a loop and a helix. This stretch is in the open conformation when the enzyme is in the free state as in the crystals of M. tuberculosis peptidyl-tRNA hydrolase. Furthermore, there is no physical continuity between the tRNA and the peptide-binding sites. The molecule in the E. coli crystal mimics the peptide-bound enzyme molecule. The peptide stretch referred to earlier now closes on the bound peptide. Concurrently, a channel connecting the tRNA and the peptide-binding site opens primarily through the concerted movement of two residues. Thus, the crystal structure of M. tuberculosis peptidyl-tRNA hydrolase when compared with the crystal structure of the E. coli enzyme, leads to a model of structural changes associated with enzyme action on the basis of the plasticity of the molecule. (c) 2007 Elsevier Ltd. All rights reserved.

Investigations of the scaling criteria for a mild combustion burner

In this paper, a new strategy for scaling burners based on "mild combustion" is evolved and adopted to scaling a burner from 3 to a 150 kW burner at a high heat release Late of 5 MW/m(3) Existing scaling methods (constant velocity, constant residence time, and Cole's procedure [Proc. Combust. Inst. 28 (2000) 1297]) are found to be inadequate for mild combustion burners. Constant velocity approach leads to reduced heat release rates at large sizes and constant residence time approach in unacceptable levels of pressure drop across the system. To achieve mild combustion at high heat release rates at all scales, a modified approach with high recirculation is adopted in the present studies. Major geometrical dimensions are scaled as D similar to Q(1/3) with an air injection velocity of similar to 100 m/s (Delta p similar to 600 mm water gauge). Using CFD support, the position of air injection holes is selected to enhance the recirculation rates. The precise role of secondary air is to increase the recirculation rates and burn LIP the residual CO in the downstream. Measurements of temperature and oxidizer concentrations inside 3 kW, 150 kW burner and a jet flame are used to distinguish the combustion process in these burners. The burner can be used for a wide range of fuels from LPG to producer gas as extremes. Up to 8 dB of noise level reduction is observed in comparison to the conventional combustion mode. Exhaust NO emissions below 26 and 3 ppm and temperatures 1710 and 1520 K were measured for LPG and producer gas when the burner is operated at stoichiometry. (c) 2004 The Combustion Institute. Published by Elsevier Inc. All rights reserved.

Structural plasticity and enzyme action: Crystal structures of Mycobacterium tuberculosis peptidyl-tRNA hydrolase

Peptidyl-tRNA hydrolase cleaves the ester bond between tRNA and the attached peptide in peptidyl-tRNA in order to avoid the toxicity resulting from its accumulation and to free the tRNA available for further rounds in protein synthesis. The structure of the enzyme from Mycobacteritan tuberculosis has been determined in three crystal forms. This structure and the structure of the enzyme frorn Escherichia coli in its crystal differ substantially on account of the binding of the C terminus of the E. coli enzyme to the peptide-binding site of a neighboring molecule in the crystal. A detailed examination of this difference led to an elucidation of the plasticity of the binding site of the enzyme. The peptide-binding site of the enzyme is a cleft between the body, of the molecule and a polypepticle Y stretch involving a loop and a helix. This stretch is in the open conformation when the enzyme is in the free state as in the crystals of M. tuberculosis peptidyl-tRNA hydrolase. Furthermore, there is no physical continuity between the tRNA and the peptide-binding sites. The molecule in the E. coli crystal mimics the peptide-bound enzyme molecule. The peptide stretch referred to earlier now closes on the bound peptide. Concurrently, a channel connecting the tRNA and the peptide-binding site opens primarily through the concerted movement of two residues. Thus, the crystal structure of M. tuberculosis peptidyl-tRNA hydrolase when compared with the crystal structure of the E. coli enzyme, leads to a model of structural changes associated with enzyme action on the basis of the plasticity of the molecule. (c) 2007 Elsevier Ltd. All rights reserved.

Chemical approaches to penicillin allergy—IV. Binding of carrier receptor protein with penicillin and its analogues

The availability of electrophoretically homogeneous rabbit penicillin carrier receptor protein (CRP) by affinity chromatography afforded an idealin vitro system to calculate the thermodynamic parameters of binding of penicillin and analogues with CRP as well as competitive binding of such analogues with CRP in presence of14C-penicillin G. The kinetics of association of CRP with 7-deoxy penicillin which does not bind covalently with CRP have been studied through equilibrium dialysis with14C-7-deoxybenzyl penicillin and found to be K=2·79×106M−1.−ΔG=8·106 k cal/mole as well as fluorescence quenching studies with exciter λ 280 K=3·573×106M−1,−ΔG=8·239 k cal/mole. The fluorescence quenching studies have been extended to CRP-benzyl penicillin and CRP-6-aminopenicillanic acid (6APA) systems also. The fluorescence data with benzyl penicillin indicate two conformational changes in CRP—a fast change corresponding to the non-covalent binding to CRP with 7-deoxy penicillin and a slower change due to covalent bond formation. With 6-APA the first change is not observed but the conformational change corresponding to covalent binding is only seen. Competitive binding studies indicate that the order of binding of CRP with the analogues of penicillin is as follows: methicillin > 6APA > carbenicillin >o-nitrobenzyl penicillin > cloxacillin ≈ benzyl penicillin ≈ 6-phenyl acetamido penicillanyl alcohol ≈ 7 phenyl acetamido desacetoxy cephalosporanic acid ≈p-amino benzyl penicillin ≈p-nitro benzyl penicillin > ticarcillin >o-amino benzyl penicillin > amoxycillin > 7-deoxy benzyl penicillin > ampicillin.From these data it has been possible to delineate partially the topology of the penicillin binding cleft of the CRP as well as some of the functional groups in the cleft responsible for the binding process.

The behaviour of excited plane jets

A plane subsonic jet can be excited to entrain more fluid from its surroundings by subjecting it to antisymmetric periodic disturbances. The essential feature in this phenomenon is the rolling-up motion of an initially flapping jet to form large vortices which are responsible for greater entrainment. Several methods developed to impart oscillations to the flow at the nozzle, such as the acoustic pressure oscillator, the vibration of a single vane in the potential core region, the reciprocating lip system and the twin vane exciter, are described in this article. A minimum threshold in amplitude is necessary for exciting the flow. However, the frequency of oscillation is much less than that predicted by stability considerations.

Protein Hydration and Water Structure: X-ray Analysis of a Closely Packed Protein Crystal with Very Low Solvent Content

Low-humidity monoclinic lysozyme, resulting from a water-mediated transformation, has one of the lowest solvent contents (22% by volume) observed in a protein crystal. Its structure has been solved by the molecular replacement method and refined to an R value of 0.175 for 7684 observed reflections in the 10–1.75 Å resolution shell. 90% of the solvent in the well ordered crystals could be located. Favourable sites of hydration on the protein surface include side chains with multiple hydrogen-bonding centres, and regions between short hydrophilic side chains and the main-chain CO or NH groups of the same or nearby residues. Major secondary structural features are not disrupted by hydration. However, the free CO groups at the C terminii and, to a lesser extent, the NH groups at the N terminii of helices provide favourable sites for water interactions, as do reverse turns and regions which connect β-structure and helices. The hydration shell consists of discontinuous networks of water molecules, the maximum number of molecules in a network being ten. The substrate-binding cleft is heavily hydrated, as is the main loop region which is stabilized by water interactions. The protein molecules are close packed in the crystals with a molecular coordination number of 14. Arginyl residues are extensively involved in intermolecular hydrogen bonds and water bridges. The water molecules in the crystal are organized into discrete clusters. A distinctive feature of the clusters is the frequent occurrence of three-membered rings. The protein molecules undergo substantial rearrangement during the transformation from the native to the low-humidity form. The main-chain conformations in the two forms are nearly the same, but differences exist in the side-chain conformation. The differences are particularly pronounced in relation to Trp 62 and Trp 63. The shift in Trp 62 is especially interesting as it is also known to move during inhibitor binding.

A finite element analysis of dynamic fracture initiation by ductile failure mechanisms in a 4340 steel

In some recent dropweight impact experiments [5] with pre-notched bend specimens of 4340 steel, it was observed that considerable crack tunneling occurred in the interior of the specimen prior to gross fracture initiation on the free surfaces. The final failure of the side ligaments happened because of shear lip formation. The tunneled region is characterized by a flat, fibrous fracture surface. In this paper, the experiments of [5] (corresponding to 5 m/s impact speed) are analyzed using a plane strain, dynamic finite element procedure. The Gurson constitutive model that accounts for the ductile failure mechanisms of micro-void nucleation, growth and coalescence is employed. The time at which incipient failure was observed near the notch tip in this computation, and the value of the dynamic J-integral, J d, at this time, compare reasonably well with experiments. This investigation shows that J-controlled stress and deformation fields are established near the notch tip whenever J d , increases with time. Also, it is found that the evolution of micro-mechanical quantities near the notch root can be correlated with the time variation of J d .The strain rate and the adiabatic temperature rise experienced at the notch root are examined. Finally, spatial variations of stresses and deformations are analyzed in detail.

Identification of Critical Residues of the Mycobacterial Dephosphocoenzyme A Kinase by Site-Directed Mutagenesis

Dephosphocoenzyme A kinase performs the transfer of the c-phosphate of ATP to dephosphocoenzyme A, catalyzing the last step of coenzyme A biosynthesis. This enzyme belongs to the P-loop-containing NTP hydrolase superfamily, all members of which posses a three domain topology consisting of a CoA domain that binds the acceptor substrate, the nucleotide binding domain and the lid domain. Differences in the enzymatic organization and regulation between the human and mycobacterial counterparts, have pointed out the tubercular CoaE as a high confidence drug target (HAMAP database). Unfortunately the absence of a three-dimensional crystal structure of the enzyme, either alone or complexed with either of its substrates/regulators, leaves both the reaction mechanism unidentified and the chief players involved in substrate binding, stabilization and catalysis unknown. Based on homology modeling and sequence analysis, we chose residues in the three functional domains of the enzyme to assess their contributions to ligand binding and catalysis using site-directed mutagenesis. Systematically mutating the residues from the P-loop and the nucleotide-binding site identified Lys14 and Arg140 in ATP binding and the stabilization of the phosphoryl intermediate during the phosphotransfer reaction. Mutagenesis of Asp32 and Arg140 showed catalytic efficiencies less than 5-10% of the wild type, indicating the pivotal roles played by these residues in catalysis. Non-conservative substitution of the Leu114 residue identifies this leucine as the critical residue from the hydrophobic cleft involved in leading substrate, DCoA binding. We show that the mycobacterial enzyme requires the Mg2+ for its catalytic activity. The binding energetics of the interactions of the mutant enzymes with the substrates were characterized in terms of their enthalpic and entropic contributions by ITC, providing a complete picture of the effects of the mutations on activity. The properties of mutants defective in substrate recognition were consistent with the ordered sequential mechanism of substrate addition for CoaE.

Functional analysis of conserved motifs in EcoP15I DNA methyltransferase

EcoP15I DNA methyltransferase recognizes the sequence 5'-CAGCAG-3' and transfers a methyl group to N-6 of the second adenine residue in the recognition sequence. All N-6 adenine methyltransferases contain two highly conserved sequences, FxGxG (motif I), postulated to form part of the S-adenosyl-L-methionine binding site and (D/N/S)PP(Y/F) (motif IV) involved in catalysis. We have altered the second glycine residue in motif I to arginine and serine, and substituted tyrosine in motif IV with tryptophan in EcoP15I DNA methyltransferase, using site-directed mutagenesis. The mutant enzymes were overexpressed, purified and characterized by biochemical methods. The mutations in motif I completely abolished AdoMet binding but left target DNA recognition unaltered. Although the mutation in motif IV resulted in loss of enzyme activity, we observed enhanced crosslinking of S-adenosyl-L-methionine and DNA. This implies that DNA and AdoMet binding sites are close to motif IV. Taken together, these results reinforce the importance of motif I in AdoMet binding and motif IV in catalysis. Additionally, limited proteolysis and UV crosslinking experiments with EcoP15I DNA methyltransferase imply that DNA binds in a cleft formed by two domains in the protein. Methylation protection analysis provides evidence for the fact that EcoP15I DNA MTase makes contacts in the major groove of its substrate DNA. Interestingly, hypermethylation of the guanine residue next to the target adenine residue indicates that the protein probably flips out the target adenine residue. (C) 1996 Academic Press Limited

New triquinane-based host molecules : binding with diamines

Synthesis of several shape-specific hosts through heteroaromatic annulation on cis,syn,cis-triquinanedione 1 and X-ray crystal structure determination of one of them, 4a, is reported. Preliminary results of complexation between cleft 5a and diamines are reported.