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Sample records for antibacterial protein lysozyme

  1. Pluronic-lysozyme conjugates as anti-adhesive and antibacterial bifunctional polymers for surface coating.

    PubMed

    Muszanska, Agnieszka K; Busscher, Henk J; Herrmann, Andreas; van der Mei, Henny C; Norde, Willem

    2011-09-01

    This paper describes the preparation and characterization of polymer-protein conjugates composed of a synthetic triblock copolymer with a central polypropylene oxide (PPO) block and two terminal polyethylene oxide (PEO) segments, Pluronic F-127, and the antibacterial enzyme lysozyme attached to the telechelic groups of the PEO chains. Covalent conjugation of lysozyme proceeded via reductive amination of aldehyde functionalized PEO blocks (CHO-Pluronic) and the amine groups of the lysine residues in the protein. SDS-PAGE gel electrophoresis together with MALDI-TOF mass spectrometry analysis revealed formation of conjugates of one or two lysozyme molecules per Pluronic polymer chain. The conjugated lysozyme showed antibacterial activity towards Bacillus subtilis. Analysis with a quartz crystal microbalance with dissipation revealed that Pluronic-lysozyme conjugates adsorb in a brush conformation on a hydrophobic gold-coated quartz surface. X-ray photoelectron spectroscopy indicated surface coverage of 32% by lysozyme when adsorbed from a mixture of unconjugated Pluronic and Pluronic-lysozyme conjugate (ratio 99:1) and of 47% after adsorption of 100% Pluronic-lysozyme conjugates. Thus, bifunctional brushes were created, possessing both anti-adhesive activity due to the polymer brush, combined with the antibacterial activity of lysozyme. The coating having a lower degree of lysozyme coverage proved to be more bactericidal.

  2. Scientist prepare Lysozyme Protein Crystal

    NASA Technical Reports Server (NTRS)

    1996-01-01

    Dan Carter and Charles Sisk center a Lysozyme Protein crystal grown aboard the USML-2 shuttle mission. Protein isolated from hen egg-white and functions as a bacteriostatic enzyme by degrading bacterial cell walls. First enzyme ever characterized by protein crystallography. It is used as an excellent model system for better understanding parameters involved in microgravity crystal growth experiments. The goal is to compare kinetic data from microgravity experiments with data from laboratory experiments to study the equilibrium.

  3. Thermophysical properties of lysozyme (protein) solutions

    NASA Technical Reports Server (NTRS)

    Liu, Jiaching; Yang, Wen-Jei

    1992-01-01

    Thermophysical properties of protein solutions composed of the lysozyme crystals with a 0.1 M sodium acetate and 5 percent NaCl solution as the buffer (pH = 4.0) are determined. The properties being measured include specific heat, thermal conductivity, dynamic viscosity, and surface tension. The protein concentrations are varied. Thermal diffusivity is calculated using the measured results. The purpose of the research is to measure thermophysical properties of lysozyme solutions which would serve as the data bank for controlling and modeling the crystal growth process on earth as well as in space.

  4. Genetically Enhanced Lysozyme Evades a Pathogen Derived Inhibitory Protein

    PubMed Central

    Dostal, Sarah M.; Fang, Yongliang; Guerrette, Jonathan C.; Scanlon, Thomas C.; Griswold, Karl E.

    2015-01-01

    The accelerating spread of drug-resistant bacteria is creating demand for novel antibiotics. Bactericidal enzymes, such as human lysozyme (hLYZ), are interesting drug candidates due to their inherent catalytic nature and lack of susceptibility to the resistance mechanisms typically directed towards chemotherapeutics. However, natural antibacterial enzymes have their own limitations. For example, hLYZ is susceptible to pathogen derived inhibitory proteins, such as Escherichia coli Ivy. Here, we describe proof of concept studies demonstrating that hLYZ can be effectively redesigned to evade this potent lysozyme inhibitor. Large combinatorial libraries of hLYZ were analyzed using an innovative screening platform based on microbial co-culture in hydrogel microdroplets. Isolated hLYZ variants were orders of magnitude less susceptible to E. coli Ivy yet retained high catalytic proficiency and inherent antibacterial activity. Interestingly, the engineered escape variants showed a disadvantageous increase in susceptibility to the related Ivy ortholog from Pseudomonas aeruginosa as well as an unrelated E. coli inhibitory protein, MliC. Thus, while we have achieved our original objective with respect to escaping E. coli Ivy, engineering hLYZ for broad-spectrum evasion of proteinaceous inhibitors will require consideration of the complex and varied determinants that underlie molecular recognition by these emerging virulence factors. PMID:25607237

  5. Molecular characterization, gene structure and antibacterial activity of a g-type lysozyme from the European sea bass (Dicentrarchus labrax L.).

    PubMed

    Buonocore, Francesco; Randelli, Elisa; Trisolino, Pamela; Facchiano, Angelo; de Pascale, Donatella; Scapigliati, Giuseppe

    2014-11-01

    In fish, the first line of defense is represented by the innate immune system and the lysozyme is one of the molecules involved in this mechanism of protection. Three types of lysozymes have been identified in metazoan, the c-type (chicken or conventional), the g-type (goose-type) and the i-type (invertebrate type). They are all involved in the hydrolysation of the bacterial cell wall. Our work has been focused on the molecular characterization, expression analysis by real-time PCR, both at basal condition and after in vivo challenges, and 3D structural studies on the g-type lysozyme from sea bass (Dicentrarchus labrax L.). Moreover, a recombinant sea bass lysozyme has been produced in Escherichia coli and used to investigate the activity of the enzyme at different pH and temperatures and to perform antibacterial assays against typical fish pathogens. The cloned sea bass cDNA for g-type lysozyme (accession number FN667957) consists of 742 bp and translates for a putative protein of 188 amino acids. The molecular weight is 20.251, 41Da with a theoretical pI of 8.53, two cysteine residues along the sequence and no putative signal peptide. These features of the enzyme are in agreement with the expected characteristics of a proper g-type lysozyme, except for the cysteine residues that in fish are quite variable in number. An alignment between known g-type lysozyme sequences evidences that the amino acid residues thought to be involved in the enzyme catalysis (Glu(71), Asp(84) and Asp(95) in sea bass) are quite well conserved between mammalian, avian and fish sequences. The sea bass g-type lysozyme gene is composed of four exons and three introns and this gene structure is more compact compared to other known fish lysozyme homologues. Modeling of 3D structure has been performed on the template structure of g-type lysozyme from Atlantic cod. The catalytic site appears well conserved when compared with known structures of fish g-type lysozymes (cod and salmon). The basal

  6. Development of lysozyme-combined antibacterial system to reduce sulfur dioxide and to stabilize Italian Riesling ice wine during aging process

    PubMed Central

    Chen, Kai; Han, Shun-yu; Zhang, Bo; Li, Min; Sheng, Wen-jun

    2015-01-01

    For the purpose of SO2 reduction and stabilizing ice wine, a new antibacterial technique was developed and verified in order to reduce the content of sulfur dioxide (SO2) and simultaneously maintain protein stability during ice wine aging process. Hazardous bacterial strain (lactic acid bacteria, LAB) and protein stability of Italian Riesling ice wine were evaluated in terms of different amounts of lysozyme, SO2, polyphenols, and wine pH by single-factor experiments. Subsequently, a quadratic rotation-orthogonal composite design with four variables was conducted to establish the multiple linear regression model that demonstrated the influence of different treatments on synthesis score between LAB inhibition and protein stability of ice wine. The results showed that, synthesis score can be influenced by lysozyme and SO2 concentrations on an extremely significant level (P < 0.01). Furthermore, the lysozyme-combined antibacterial system, which is specially designed for ice wine aging, was optimized step by step by response surface methodology and ridge analysis. As a result, the optimal proportion should be control in ice wine as follows: 179.31 mg L−1 lysozyme, 177.14 mg L−1 SO2, 0.60 g L−1 polyphenols, and 4.01 ice wine pH. Based on this system, the normalized synthesis score between LAB inhibition and protein stability can reach the highest point 0.920. Finally, by the experiments of verification and comparison, it was indicated that lysozyme-combined antibacterial system, which was a practical and prospective method to reduce SO2 concentration and effectively prevent contamination from hazardous LAB, can be used to stabilize ice wine during aging process. PMID:26405531

  7. Molecular cloning, inducible expression and antibacterial analysis of a novel i-type lysozyme (lyz-i2) in Pacific white shrimp, Litopenaeus vannamei.

    PubMed

    Chen, Ting; Ren, Chunhua; Wang, Yanhong; Luo, Peng; Jiang, Xiao; Huang, Wen; Chen, Chang; Hu, Chaoqun

    2016-07-01

    The full-length cDNA coding for a novel invertebrate (i-type) lysozyme was identified in Pacific white shrimp (Litopenaeus vannamei). The newly obtained L. vannamei lysozyme is similar to the Penaeus monodon i-type lysozyme 2, but it is distant from the known L. vannamei c-type lysozyme and i-type lysozyme 1 in protein sequence; therefore, it was defined as L. vannamei i-type lysozyme 2 (lyz-i2). Expression of L. vannamei lyz-i2 transcripts were ubiquitously detected in all tissues we selected, with the highest abundance observed in the hemolymph. Challenge with Vibrio harveyi might elicit L. vannamei lyz-i2 mRNA expression in the hepatopancreas, intestine, muscle, gill and hemolymph. In the themolymph, specifically, the stimulatory effects of Vibrio and lipopolysaccharide (LPS) on lyz-i2 transcript levels were durable and transient, respectively; while Polyinosinic:polycytidylic acid [Poly (I:C)] treatment did not affect lyz-i2 expression. L. vannamei lyz-i2 recombinant protein was generated in an Escherichia coli system. By lysoplate and turbidimetric assays, the L. vannamei lyz-i2 recombinant protein showed a broad spectrum of antimicrobial properties with high activities against Micrococcaceae lysodeikticus and various Vibrio species and relatively low activity against E. coli. In conclusion, L. vannamei lyz-i2 might be a potent antibacterial protein with a role in innate immunity in Penaeid shrimp.

  8. Lysozyme-coated silver nanoparticles for differentiating bacterial strains on the basis of antibacterial activity

    NASA Astrophysics Data System (ADS)

    Ashraf, Sumaira; Chatha, Mariyam Asghar; Ejaz, Wardah; Janjua, Hussnain Ahmed; Hussain, Irshad

    2014-10-01

    Lysozyme, an antibacterial enzyme, was used as a stabilizing ligand for the synthesis of fairly uniform silver nanoparticles adopting various strategies. The synthesized particles were characterized using UV-visible spectroscopy, FTIR, dynamic light scattering (DLS), and TEM to observe their morphology and surface chemistry. The silver nanoparticles were evaluated for their antimicrobial activity against several bacterial species and various bacterial strains within the same species. The cationic silver nanoparticles were found to be more effective against Pseudomonas aeruginosa 3 compared to other bacterial species/strains investigated. Some of the bacterial strains of the same species showed variable antibacterial activity. The difference in antimicrobial activity of these particles has led to the conclusion that antimicrobial products formed from silver nanoparticles may not be equally effective against all the bacteria. This difference in the antibacterial activity of silver nanoparticles for different bacterial strains from the same species may be due to the genome islands that are acquired through horizontal gene transfer (HGT). These genome islands are expected to possess some genes that may encode enzymes to resist the antimicrobial activity of silver nanoparticles. These silver nanoparticles may thus also be used to differentiate some bacterial strains within the same species due to variable silver resistance of these variants, which may not possible by simple biochemical tests.

  9. Lysozyme-coated silver nanoparticles for differentiating bacterial strains on the basis of antibacterial activity

    PubMed Central

    2014-01-01

    Lysozyme, an antibacterial enzyme, was used as a stabilizing ligand for the synthesis of fairly uniform silver nanoparticles adopting various strategies. The synthesized particles were characterized using UV-visible spectroscopy, FTIR, dynamic light scattering (DLS), and TEM to observe their morphology and surface chemistry. The silver nanoparticles were evaluated for their antimicrobial activity against several bacterial species and various bacterial strains within the same species. The cationic silver nanoparticles were found to be more effective against Pseudomonas aeruginosa 3 compared to other bacterial species/strains investigated. Some of the bacterial strains of the same species showed variable antibacterial activity. The difference in antimicrobial activity of these particles has led to the conclusion that antimicrobial products formed from silver nanoparticles may not be equally effective against all the bacteria. This difference in the antibacterial activity of silver nanoparticles for different bacterial strains from the same species may be due to the genome islands that are acquired through horizontal gene transfer (HGT). These genome islands are expected to possess some genes that may encode enzymes to resist the antimicrobial activity of silver nanoparticles. These silver nanoparticles may thus also be used to differentiate some bacterial strains within the same species due to variable silver resistance of these variants, which may not possible by simple biochemical tests. PMID:25435831

  10. In Silico and Biochemical Characterization of Lysozyme-Like Proteins in the Rat

    PubMed Central

    Narmadha, Ganapathy; Yenugu, Suresh

    2016-01-01

    Background Spermatogenesis and sperm maturation in the male reproductive tract is dictated by a variety of proteins secreted in the testis and epididymis. Though the proteome of these tissues is known, the functional role of many of these proteins remains uncharacterized. In this study, we characterize the rat Lysozyme-like (Lyzl) genes and proteins. Methods In silico tools were used to predict the primary, secondary and tertiary structures. Reverse transcription PCR, immunofluorescence and immunoblotting were used to determine the expression pattern. Lysozyme like enzyme activity was assessed by standard assays. Results Six rat Lyzl genes namely Lyzl1, Lyzl3, Lyzl4, Lyzl5, Lyzl6 and Lyzl7 were found to be highly conserved among the vertebrates with higher homology to mouse counterparts than with human counterparts. All the LYZL proteins contained the characteristic 4 disulfide bridges similar to c-type lysozyme. Only LYZL 1 and 6, conserved the active site amino acids of the lysozyme. Molecular modeling studies indicated that LYZL proteins exhibit strikingly similar three-dimensional structures among themselves. The secondary structure analysis of the recombinant LYZL proteins indicated the presence of α-helix, β-sheet and random coil with α-helix being the majority. Docking studies indicated the peptidoglycan binding nature of LYZL proteins. All the rat Lyzl mRNA transcripts (Lyzl1, Lyzl3, Lyzl4, Lyzl5, Lyzl6 and Lyzl7) are predominantly expressed in testes though some of them are expressed in tissues other than reproductive tract. Their expression was androgen independent. The rat LYZL proteins are localized in the germinal epithelium and on the spermatozoa. Recombinant LYZL1 and 6 possessed muramidase, isopeptidase and antibacterial activities. The mechanism of antibacterial action of LYZL1 and LYZL6 involved bacterial membrane damage and leakage of cellular contents. Only LYZL1 and 6 possess peptidoglycan binding ability, whereas LYZL3, LYZL4 and LYZL5

  11. The Effect of Protein Impurities on Lysozyme Crystal Growth

    NASA Technical Reports Server (NTRS)

    Judge, Russell A.; Forsythe, Elizabeth L.; Pusey, Marc L.

    1998-01-01

    While bulk crystallization from impure solutions is used industrially as a purification step for a wide variety of materials, it is a technique that has rarely been used for proteins. Proteins have a reputation for being difficult to crystallize and high purity of the initial crystallization solution is considered paramount for success in the crystallization. Although little is written on the purifying capability of protein crystallization or of the effect of impurities on the various aspects of the crystallization process, recent published reports show that crystallization shows promise and feasibility as a purification technique for proteins. In order to further examine the issue of purity in macromolecule crystallization this study investigates the effect of the protein impurities, avidin, ovalbumin and conalbumin, at concentrations up to 50%, on the solubility, crystal face growth rates and crystal purity, of the protein lysozyme. Solubility was measured in batch experiments while a computer controlled video microscope system was used to measure the f {101} and {101} lysozyme crystal face growth rates. While little effect was observed on solubility and high crystal purity was obtained (>99.99%), the effect of the impurities on the face growth rates varied from no effect to a significant face specific effect leading to growth cessation, a phenomenon that is frequently observed in protein crystal growth. The results shed interesting light on the effect of protein impurities on protein crystal growth and strengthen the feasibility of using crystallization as a unit operation for protein purification.

  12. Protein crystal growth - Growth kinetics for tetragonal lysozyme crystals

    NASA Technical Reports Server (NTRS)

    Pusey, M. L.; Snyder, R. S.; Naumann, R.

    1986-01-01

    Results are reported from theoretical and experimental studies of the growth rate of lysozyme as a function of diffusion in earth-gravity conditions. The investigations were carried out to form a comparison database for future studies of protein crystal growth in the microgravity environment of space. A diffusion-convection model is presented for predicting crystal growth rates in the presence of solutal concentration gradients. Techniques used to grow and monitor the growth of hen egg white lysozyme are detailed. The model calculations and experiment data are employed to discuss the effects of transport and interfacial kinetics in the growth of the crystals, which gradually diminished the free energy in the growth solution. Density gradient-driven convection, caused by presence of the gravity field, was a limiting factor in the growth rate.

  13. Antibacterial activity of hen egg white lysozyme against Listeria monocytogenes Scott A in foods.

    PubMed Central

    Hughey, V L; Wilger, P A; Johnson, E A

    1989-01-01

    Egg white lysozyme killed or prevented growth of Listeria monocytogenes Scott A in several foods. Lysozyme was more active in vegetables than in animal-derived foods that we tested. For maximum activity in certain foods, EDTA was required in addition to lysozyme. Lysozyme with EDTA effectively killed inoculated populations of 10(4) L. monocytogenes per g in fresh corn, fresh green beans, shredded cabbage, shredded lettuce, and carrots during storage at 5 degrees C. Control incubations without lysozyme supported growth of L. monocytogenes to 10(6) to 10(7)/g. Lysozyme had less activity in animal-derived foods, including fresh pork sausage (bratwurst) and Camembert cheese. In bratwurst, lysozyme with EDTA prevented L. monocytogenes from growing for 2 to 3 weeks but did not kill significant numbers of cells and did not prevent eventual growth. The control sausages not containing lysozyme supported rapid and heavy growth, which indicated that lysozyme was bacteriostatic for 2 to 3 weeks in fresh pork sausage. We also prepared Camembert cheese containing 10(4) L. monocytogenes cells per g and investigated the changes during ripening in cheeses supplemented with lysozyme and EDTA. Cheeses with lysozyme by itself or together with EDTA reduced the L. monocytogenes population by approximately 10-fold over the first 3 to 4 weeks of ripening. In the same period, the control cheese wheels without added lysozyme with and without chelator slowly started to grown and eventually reached 10(6) to 10(7) CFU/g after 55 days of ripening.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2494938

  14. Lysozyme-Mediated Formation of Protein-Silica Nano-Composites for Biosensing Applications (Postprint)

    DTIC Science & Technology

    2009-05-05

    reagents Lysozyme (from hen egg white ), tetramethyl orthosilicate (TMOS) and tetraethyl orthosilicate (TEOS) were purchased from Sigma–Aldrich (St. Louis...AFRL-RX-TY-TP-2009-4611 LYSOZYME -MEDIATED FORMATION OF PROTEIN-SILICA NANO-COMPOSITES FOR BIOSENSING APPLICATIONS (POSTPRINT) Madhumati...Include area code) 15-MAR-2009 Journal Article - POSTPRINT 01-MAR-2008 -- 01-MAR-2009 Lysozyme -Mediated Formation of Protein–Silica Nano-Composites for

  15. Expression of lysozymes from Erwinia amylovora phages and Erwinia genomes and inhibition by a bacterial protein.

    PubMed

    Müller, Ina; Gernold, Marina; Schneider, Bernd; Geider, Klaus

    2012-01-01

    Genes coding for lysozyme-inhibiting proteins (Ivy) were cloned from the chromosomes of the plant pathogens Erwinia amylovora and Erwinia pyrifoliae. The product interfered not only with activity of hen egg white lysozyme, but also with an enzyme from E. amylovora phage ΦEa1h. We have expressed lysozyme genes from the genomes of three Erwinia species in Escherichia coli. The lysozymes expressed from genes of the E. amylovora phages ΦEa104 and ΦEa116, Erwinia chromosomes and Arabidopsis thaliana were not affected by Ivy. The enzyme from bacteriophage ΦEa1h was fused at the N- or C-terminus to other peptides. Compared to the intact lysozyme, a His-tag reduced its lytic activity about 10-fold and larger fusion proteins abolished activity completely. Specific protease cleavage restored lysozyme activity of a GST-fusion. The bacteriophage-encoded lysozymes were more active than the enzymes from bacterial chromosomes. Viral lyz genes were inserted into a broad-host range vector, and transfer to E. amylovora inhibited cell growth. Inserted in the yeast Pichia pastoris, the ΦEa1h-lysozyme was secreted and also inhibited by Ivy. Here we describe expression of unrelated cloned 'silent' lyz genes from Erwinia chromosomes and a novel interference of bacterial Ivy proteins with a viral lysozyme.

  16. Analysis of Two Lysozyme Genes and Antimicrobial Functions of Their Recombinant Proteins in Asian Seabass

    PubMed Central

    Fu, Gui Hong; Bai, Zhi Yi; Xia, Jun Hong; Liu, Feng; Liu, Peng; Yue, Gen Hua

    2013-01-01

    Lysozymes are important proteins of the innate immune system for the defense against bacterial infection. We cloned and analyzed chicken-type (c-type) and goose-type (g-type) lysozymes from Asian seabass (Lates calcarifer). The deduced amino acid sequence of the c-type lysozyme contained 144 residues and possessed typical structure residues, conserved catalytic residues (Glu50 and Asp67) and a “GSTDYGIFQINS” motif. The deduced g-type lysozyme contained 187 residues and possessed a goose egg white lysozyme (GEWL) domain containing three conserved catalytic residues (Glu71, Asp84, Asp95) essential for catalytic activity. Real time quantitative PCR (qRT-PCR) revealed that the two lysozyme genes were constitutively expressed in all the examined tissues. The c-type lysozyme was most abundant in liver, while the g-type lysozyme was predominantly expressed in intestine and weakly expressed in muscle. The c-type and g-type transcripts were up-regulated in the kidney, spleen and liver in response to a challenge with Vibrio harveyi. The up-regulation of the c-type lysozyme was much stronger than that of the g-type lysozyme in kidney and spleen. The recombinant proteins of the c-type and g-type lysozymes showed lytic activities against the bacterial pathogens Vibrio harveyi and Photobacterium damselae in a dosage-dependent manner. We identified single nucleotide polymorphisms (SNPs) in the two lysozyme genes. There were significant associations of these polymorphisms with resistance to the big belly disease. These results suggest that the c- and g-type genes play an important role in resistance to bacterial pathogens in fish. The SNP markers in the two genes associated with the resistance to bacterial pathogens may facilitate the selection of Asian seabass resistant to bacterial diseases. PMID:24244553

  17. Scaling and self-organized criticality in proteins: Lysozyme c

    NASA Astrophysics Data System (ADS)

    Phillips, J. C.

    2009-11-01

    Proteins appear to be the most dramatic natural example of self-organized criticality (SOC), a concept that explains many otherwise apparently unlikely phenomena. Protein functionality is often dominated by long-range hydro(phobic/philic) interactions, which both drive protein compaction and mediate protein-protein interactions. In contrast to previous reductionist short-range hydrophobicity scales, the holistic Moret-Zebende hydrophobicity scale [Phys. Rev. E 75, 011920 (2007)] represents a hydroanalytic tool that bioinformatically quantifies SOC in a way fully compatible with evolution. Hydroprofiling identifies chemical trends in the activities and substrate binding abilities of model enzymes and antibiotic animal lysozymes c , as well as defensins, which have been the subject of tens of thousands of experimental studies. The analysis is simple and easily performed and immediately yields insights not obtainable by traditional methods based on short-range real-space interactions, as described either by classical force fields used in molecular-dynamics simulations, or hydrophobicity scales based on transference energies from water to organic solvents or solvent-accessible areas.

  18. Complex coacervates of hyaluronic acid and lysozyme: effect on protein structure and physical stability.

    PubMed

    Water, Jorrit J; Schack, Malthe M; Velazquez-Campoy, Adrian; Maltesen, Morten J; van de Weert, Marco; Jorgensen, Lene

    2014-10-01

    Complex coacervates of hyaluronic acid and lysozyme, a model protein, were formed by ionic interaction using bulk mixing and were characterized in terms of binding stoichiometry and protein structure and stability. The complexes were formed at pH 7.2 at low ionic strength (6mM) and the binding stoichiometry was determined using solution depletion and isothermal titration calorimetry. The binding stoichiometry of lysozyme to hyaluronic acid (870 kDa) determined by solution depletion was found to be 225.9 ± 6.6 mol, or 0.1 bound lysozyme molecules per hyaluronic acid monomer. This corresponded well with that obtained by isothermal titration calorimetry of 0.09 bound lysozyme molecules per hyaluronic acid monomer. The complexation did not alter the secondary structure of lysozyme measured by Fourier-transform infrared spectroscopy overlap analysis and had no significant impact on the Tm of lysozyme determined by differential scanning calorimetry. Furthermore, the protein stability of lysozyme was found to be improved upon complexation during a 12-weeks storage study at room temperature, as shown by a significant increase in recovered protein when complexed (94 ± 2% and 102 ± 5% depending on the polymer-protein weight to weight ratio) compared to 89 ± 2% recovery for uncomplexed protein. This study shows the potential of hyaluronic acid to be used in combination with complex coacervation to increase the physical stability of pharmaceutical protein formulations.

  19. Structure and evolution of the Ivy protein family, unexpected lysozyme inhibitors in Gram-negative bacteria

    PubMed Central

    Abergel, Chantal; Monchois, Vincent; Byrne, Deborah; Chenivesse, Sabine; Lembo, Frédérique; Lazzaroni, Jean-Claude; Claverie, Jean-Michel

    2007-01-01

    Part of an ancestral bactericidal system, vertebrate C-type lysozyme targets the peptidoglycan moiety of bacterial cell walls. We report the crystal structure of a protein inhibitor of C-type lysozyme, the Escherichia coli Ivy protein, alone and in complex with hen egg white lysozyme. Ivy exhibits a novel fold in which a protruding five-residue loop appears essential to its inhibitory effect. This feature guided the identification of Ivy orthologues in other Gram-negative bacteria. The structure of the evolutionary distant Pseudomonas aeruginosa Ivy orthologue was also determined in complex with hen egg white lysozyme, and its antilysozyme activity was confirmed. Ivy expression protects porous cell-wall E. coli mutants from the lytic effect of lysozyme, suggesting that it is a response against the permeabilizing effects of the innate vertebrate immune system. As such, Ivy acts as a virulence factor for a number of Gram-negative bacteria-infecting vertebrates. PMID:17405861

  20. Binding of lysozyme to phospholipid bilayers: evidence for protein aggregation upon membrane association.

    PubMed

    Gorbenko, Galyna P; Ioffe, Valeriya M; Kinnunen, Paavo K J

    2007-07-01

    Biological functions of lysozyme, including its antimicrobial, antitumor, and immune-modulatory activities have been suggested to be largely determined by the lipid binding properties of this protein. To gain further insight into these interactions on a molecular level the association of lysozyme to liposomes composed of either 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine or its mixtures with 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-phosphatidylserine, or bovine heart cardiolipin was studied by a combination of fluorescence techniques. The characteristics of the adsorption of lysozyme to lipid bilayers were investigated using fluorescein 5'-isothiocyanate labeled protein, responding to membrane association by a decrease in fluorescence. Upon increasing the content of anionic phospholipids in lipid vesicles, the binding isotherms changed from Langmuir-like to sigmoidal. Using adsorption models based on scaled particle and double-layer theories, this finding was rationalized in terms of self-association of the membrane-bound protein. The extent of quenching of lysozyme tryptophan fluorescence by acrylamide decreased upon membrane binding, revealing a conformational transition for the protein upon its surface association, resulting in a diminished access of the fluorophore to the aqueous phase. Steady-state fluorescence anisotropy of bilayer-incorporated probe 1,6-diphenyl-1,3,5-hexatriene was measured at varying lipid-to-protein molar ratios. Lysozyme was found to increase acyl-chain order for liposomes with the content of acidic phospholipid exceeding 10 mol %. Both electrostatic and hydrophobic protein-lipid interactions can be concluded to modulate the aggregation behavior of lysozyme when bound to lipid bilayers. Modulation of lysozyme aggregation propensity by membrane binding may have important implications for protein fibrillogenesis in vivo. Disruption of membrane integrity by the aggregated

  1. Binding of Lysozyme to Phospholipid Bilayers: Evidence for Protein Aggregation upon Membrane Association

    PubMed Central

    Gorbenko, Galyna P.; Ioffe, Valeriya M.; Kinnunen, Paavo K. J.

    2007-01-01

    Biological functions of lysozyme, including its antimicrobial, antitumor, and immune-modulatory activities have been suggested to be largely determined by the lipid binding properties of this protein. To gain further insight into these interactions on a molecular level the association of lysozyme to liposomes composed of either 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine or its mixtures with 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-phosphatidylserine, or bovine heart cardiolipin was studied by a combination of fluorescence techniques. The characteristics of the adsorption of lysozyme to lipid bilayers were investigated using fluorescein 5′-isothiocyanate labeled protein, responding to membrane association by a decrease in fluorescence. Upon increasing the content of anionic phospholipids in lipid vesicles, the binding isotherms changed from Langmuir-like to sigmoidal. Using adsorption models based on scaled particle and double-layer theories, this finding was rationalized in terms of self-association of the membrane-bound protein. The extent of quenching of lysozyme tryptophan fluorescence by acrylamide decreased upon membrane binding, revealing a conformational transition for the protein upon its surface association, resulting in a diminished access of the fluorophore to the aqueous phase. Steady-state fluorescence anisotropy of bilayer-incorporated probe 1,6-diphenyl-1,3,5-hexatriene was measured at varying lipid-to-protein molar ratios. Lysozyme was found to increase acyl-chain order for liposomes with the content of acidic phospholipid exceeding 10 mol %. Both electrostatic and hydrophobic protein-lipid interactions can be concluded to modulate the aggregation behavior of lysozyme when bound to lipid bilayers. Modulation of lysozyme aggregation propensity by membrane binding may have important implications for protein fibrillogenesis in vivo. Disruption of membrane integrity by the aggregated

  2. Lysozyme-lysozyme self-interactions as assessed by the osmotic second virial coefficient: impact for physical protein stabilization.

    PubMed

    Le Brun, Virginie; Friess, Wolfgang; Schultz-Fademrecht, Torsten; Muehlau, Silke; Garidel, Patrick

    2009-09-01

    The purpose of the presented study is to understand the physicochemical properties of proteins in aqueous solutions in order to identify solution conditions with reduced attractive protein-protein interactions, to avoid the formation of protein aggregates and to increase protein solubility. This is assessed by measuring the osmotic second virial coefficient (B(22)), a parameter of solution non-ideality, which is obtained using self-interaction chromatography. The model protein is lysozyme. The influence of various solution conditions on B(22) was investigated: protonation degree, ionic strength, pharmaceutical relevant excipients and combinations thereof. Under acidic solution conditions B(22) is positive, favoring protein repulsion. A similar trend is observed for the variation of the NaCl concentration, showing that with increasing the ionic strength protein attraction is more likely. B(22) decreases and becomes negative. Thus, solution conditions are obtained favoring attractive protein-protein interactions. The B(22) parameter also reflects, in general, the influence of the salts of the Hofmeister series with regard to their salting-in/salting-out effect. It is also shown that B(22) correlates with protein solubility as well as physical protein stability.

  3. Growth of gold nanoclusters and nanocrystals induced by lysozyme protein in thin film conformation

    NASA Astrophysics Data System (ADS)

    Bhowal, Ashim Chandra; Kundu, Sarathi

    2016-08-01

    Structures and growth behavior of gold nanoclusters and nanocrystals have been explored on thin films of globular protein lysozyme by using UV-vis and photoluminescence spectroscopy, X-ray diffraction (XRD) and atomic force microscopy (AFM). A simple and one-step environment friendly method has been used to grow nanocrystals on protein surface from HAuCl4 solution. It has been found that if different interaction times are provided between lysozyme films and HAuCl4 solution, then initially formed tiny gold nanoclusters on protein surface transform into nanocrystals with the passage of time. XRD analysis shows the formation of faced-centered cubic lattice along (1 1 1) crystalline direction and AFM images confirm the presence of circular, rod-like, triangular and hexagonal crystal structures. Langmuir-like growth behavior has been identified for both the gold nanoclusters and nanocrystals formation induced by the lysozyme films, however, nanocrystal growth is relatively slower than nanocluster.

  4. The effect of protein contaminants on the crystallization of turkey egg white lysozyme

    NASA Astrophysics Data System (ADS)

    Abergel, Chantal; Nesa, Marie P.; Fontecilla-Camps, Juan C.

    1991-03-01

    We report here a series of studies on the controlled contamination of crystallizing solutions of the hexagonal form of turkey egg white lysozyme (TEWL) carried out to understand the effects of impurities on the nucleation and growth of protein crystals. The contamination of TEWL solutions with any of three other avian lysozymes affects both the nucleation and the growth processes. For hen and quail egg white lysozymes, low and medium levels of contamination result in partial inhibition of nucleation and shortening of the c-axis. Further increase of the contaminant concentration leads to detectable co-crystallization. A different effect is obtained when using the pheasant egg white lysozyme. Contamination by an unrelated protein, ribonuclease A, has an effect on the nucleation levels that is similar to those observed with the avian lysozymes. However, no effect on TEWL crystal morphology is observed. Thus, in the case of TEWL crystals, one can distinguish between a specific effect on crystal morphology induced by related proteins and a more general inhibitory effect on the nucleation levels observed in all cases studied here.

  5. Characterizing protein activities on the lysozyme and nanodiamond complex prepared for bio applications.

    PubMed

    Perevedentseva, E; Cai, P-J; Chiu, Y-C; Cheng, C-L

    2011-02-01

    Recently, nanodiamond particles have attracted increasing attention as a promising nanomaterial for its biocompatibility, easy functionalization and conjugation with biomolecules, and its superb physical/chemical properties. Nanodiamonds are mainly used as markers for cell imaging, using its fluorescence or Raman signals for detection, and as carriers for drug delivery. For the success of these applications, the biomolecule associated with the nanodiamond has to retain its functionality. In this work, the protein activities of egg white lysozyme adsorbed on nanodiamond particles of different sizes is investigated. The lysozyme nanodiamond complex is used here as a protein model for analyzing its structural conformation changes and, correspondingly, its enzymatic activity after the adsorption. Fourier-transform infrared spectroscopy (FTIR) is used for the analysis of the sensitive protein secondary structure. To access the activities of the adsorbed lysozyme, a fluorescence-based assay is used. The process of adsorption is also analyzed using UV-visible spectroscopic measurements in combination with analysis of nanodiamond properties with FTIR, Raman spectroscopy, and ζ-potential measurements. It is found that the activity of lysozyme upon adsorption depends on the nanodiamond's size and surface properties, and that the nanodiamond particles can be selected and treated, which do not alter the lysozyme functional properties. Such nanodiamonds can be considered convenient nanoparticles for various bioapplications.

  6. Effective protein-protein interaction from structure factor data of a lysozyme solution

    SciTech Connect

    Abramo, M. C.; Caccamo, C.; Costa, D.; Ruberto, R.; Wanderlingh, U.; Cavero, M.; Pellicane, G.

    2013-08-07

    We report the determination of an effective protein-protein central potential for a lysozyme solution, obtained from the direct inversion of the total structure factor of the system, as extracted from small angle neutron scattering. The inversion scheme rests on a hypernetted-chain relationship between the effective potential and the structural functions, and is preliminarily tested for the case of a Lennard-Jones interaction. The characteristics of our potential are discussed in comparison with current models of effective interactions in complex fluids. The phase behavior predictions are also investigated.

  7. Establishing Antibacterial Multilayer Films on the Surface of Direct Metal Laser Sintered Titanium Primed with Phase-Transited Lysozyme

    PubMed Central

    Guan, Binbin; Wang, Haorong; Xu, Ruiqing; Zheng, Guoying; Yang, Jie; Liu, Zihao; Cao, Man; Wu, Mingyao; Song, Jinhua; Li, Neng; Li, Ting; Cai, Qing; Yang, Xiaoping; Li, Yanqiu; Zhang, Xu

    2016-01-01

    Direct metal laser sintering is a technology that allows the fabrication of titanium (Ti) implants with a functional gradation of porosity and surface roughness according to three-dimensional (3D) computer data. The surface roughness of direct metal laser sintered titanium (DMLS-Ti) implants may provide abundant binding sites for bacteria. Bacterial colonization and subsequent biofilm formation can cause unsatisfactory cell adhesion and implant-related infections. To prevent such infections, a novel phase-transited lysozyme (PTL) was utilized as an initial functional layer to simply and effectively prime DMLS-Ti surfaces for subsequent coating with antibacterial multilayers. The purpose of the present study was to establish a surface with dual biological functionality. The minocycline-loaded polyelectrolyte multilayers of hyaluronic acid (HA) and chitosan (CS) formed via a layer-by-layer (LbL) self-assembly technique on PTL-functionalized DMLS-Ti were designed to inhibit pathogenic microbial infections while allowing the DMLS-Ti itself and the modified coatings to retain acceptable biocompatibility. The experimental results indicate that the DMLS-Ti and the hydrogel treated surfaces can inhibit early bacterial adhesion while completely preserving osteoblast functions. This design is expected to gain considerable interest in the medical field and to have good potential for applications in multifunctional DMLS-Ti implants. PMID:27821857

  8. Establishing Antibacterial Multilayer Films on the Surface of Direct Metal Laser Sintered Titanium Primed with Phase-Transited Lysozyme

    NASA Astrophysics Data System (ADS)

    Guan, Binbin; Wang, Haorong; Xu, Ruiqing; Zheng, Guoying; Yang, Jie; Liu, Zihao; Cao, Man; Wu, Mingyao; Song, Jinhua; Li, Neng; Li, Ting; Cai, Qing; Yang, Xiaoping; Li, Yanqiu; Zhang, Xu

    2016-11-01

    Direct metal laser sintering is a technology that allows the fabrication of titanium (Ti) implants with a functional gradation of porosity and surface roughness according to three-dimensional (3D) computer data. The surface roughness of direct metal laser sintered titanium (DMLS-Ti) implants may provide abundant binding sites for bacteria. Bacterial colonization and subsequent biofilm formation can cause unsatisfactory cell adhesion and implant-related infections. To prevent such infections, a novel phase-transited lysozyme (PTL) was utilized as an initial functional layer to simply and effectively prime DMLS-Ti surfaces for subsequent coating with antibacterial multilayers. The purpose of the present study was to establish a surface with dual biological functionality. The minocycline-loaded polyelectrolyte multilayers of hyaluronic acid (HA) and chitosan (CS) formed via a layer-by-layer (LbL) self-assembly technique on PTL-functionalized DMLS-Ti were designed to inhibit pathogenic microbial infections while allowing the DMLS-Ti itself and the modified coatings to retain acceptable biocompatibility. The experimental results indicate that the DMLS-Ti and the hydrogel treated surfaces can inhibit early bacterial adhesion while completely preserving osteoblast functions. This design is expected to gain considerable interest in the medical field and to have good potential for applications in multifunctional DMLS-Ti implants.

  9. Interaction of lysozyme protein with different sized silica nanoparticles and their resultant structures

    NASA Astrophysics Data System (ADS)

    Yadav, Indresh; Aswal, V. K.; Kohlbrecher, J.

    2016-05-01

    The interaction of model protein-lysozyme with three different sized anionic silica nanoparticles has been studied by UV-vis spectroscopy, dynamic light scattering (DLS) and small-angle neutron scattering (SANS). The surface area and curvature of the nanoparticles change with size, which significantly influence their interaction with protein. The lysozyme adsorbs on the surface of the nanoparticles due to electrostatic attraction and leads to the phase transformation from one phase (clear) to two-phase (turbid) of the nanoparticle-protein system. The dominance of lysozyme induced short-range attraction over long-range electrostatic repulsion between nanoparticles is responsible for phase transformation and modeled by the two-Yukawa potential. The magnitude of the attractive interaction increases with the size of the nanoparticles as a result the phase transformation commences relatively at lower concentration of lysozyme. The structure of the nanoparticle-protein system in two-phase is characterized by the diffusion limited aggregate type of mass fractal morphology.

  10. Molecular Dynamics Analysis of Lysozyme Protein in Ethanol-Water Mixed Solvent Environment

    NASA Astrophysics Data System (ADS)

    Ochije, Henry Ikechukwu

    Effect of protein-solvent interaction on the protein structure is widely studied using both experimental and computational techniques. Despite such extensive studies molecular level understanding of proteins and some simple solvents is still not fully understood. This work focuses on detailed molecular dynamics simulations to study of solvent effect on lysozyme protein, using water, alcohol and different concentrations of water-alcohol mixtures as solvents. The lysozyme protein structure in water, alcohol and alcohol-water mixture (0-12% alcohol) was studied using GROMACS molecular dynamics simulation code. Compared to water environment, the lysozome structure showed remarkable changes in solvents with increasing alcohol concentration. In particular, significant changes were observed in the protein secondary structure involving alpha helices. The influence of alcohol on the lysozyme protein was investigated by studying thermodynamic and structural properties. With increasing ethanol concentration we observed a systematic increase in total energy, enthalpy, root mean square deviation (RMSD), and radius of gyration. a polynomial interpolation approach. Using the resulting polynomial equation, we could determine above quantities for any intermediate alcohol percentage. In order to validate this approach, we selected an intermediate ethanol percentage and carried out full MD simulation. The results from MD simulation were in reasonably good agreement with that obtained using polynomial approach. Hence, the polynomial approach based method proposed here eliminates the need for computationally intensive full MD analysis for the concentrations within the range (0-12%) studied in this work.

  11. Size-dependent interaction of silica nanoparticles with lysozyme and bovine serum albumin proteins

    NASA Astrophysics Data System (ADS)

    Yadav, Indresh; Aswal, Vinod K.; Kohlbrecher, Joachim

    2016-05-01

    The interaction of three different sized (diameter 10, 18, and 28 nm) anionic silica nanoparticles with two model proteins—cationic lysozyme [molecular weight (MW) 14.7 kDa)] and anionic bovine serum albumin (BSA) (MW 66.4 kDa) has been studied by UV-vis spectroscopy, dynamic light scattering (DLS), and small-angle neutron scattering (SANS). The adsorption behavior of proteins on the nanoparticles, measured by UV-vis spectroscopy, is found to be very different for lysozyme and BSA. Lysozyme adsorbs strongly on the nanoparticles and shows exponential behavior as a function of lysozyme concentration irrespective of the nanoparticle size. The total amount of adsorbed lysozyme, as governed by the surface-to-volume ratio, increases on lowering the size of the nanoparticles for a fixed volume fraction of the nanoparticles. On the other hand, BSA does not show any adsorption for all the different sizes of the nanoparticles. Despite having different interactions, both proteins induce similar phase behavior where the nanoparticle-protein system transforms from one phase (clear) to two phase (turbid) as a function of protein concentration. The phase behavior is modified towards the lower concentrations for both proteins with increasing the nanoparticle size. DLS suggests that the phase behavior arises as a result of the nanoparticles' aggregation on the addition of proteins. The size-dependent modifications in the interaction potential, responsible for the phase behavior, have been determined by SANS data as modeled using the two-Yukawa potential accounting for the repulsive and attractive interactions in the systems. The protein-induced interaction between the nanoparticles is found to be short-range attraction for lysozyme and long-range attraction for BSA. The magnitude of attractive interaction irrespective of protein type is enhanced with increase in the size of the nanoparticles. The total (attractive+repulsive) potential leading to two-phase formation is found to be

  12. Effects of protein and phosphate buffer concentrations on thermal denaturation of lysozyme analyzed by isoconversional method.

    PubMed

    Cao, X M; Tian, Y; Wang, Z Y; Liu, Y W; Wang, C X

    2016-07-03

    Thermal denaturation of lysozymes was studied as a function of protein concentration, phosphate buffer concentration, and scan rate using differential scanning calorimetry (DSC), which was then analyzed by the isoconversional method. The results showed that lysozyme thermal denaturation was only slightly affected by the protein concentration and scan rate. When the protein concentration and scan rate increased, the denaturation temperature (Tm) also increased accordingly. On the contrary, the Tm decreased with the increase of phosphate buffer concentration. The denaturation process of lysozymes was accelatated and the thermal stability was reduced with the increase of phosphate concentration. One part of degeneration process was not reversible where the aggregation occurred. The other part was reversible. The apparent activation energy (Ea) was computed by the isoconversional method. It decreased with the increase of the conversion ratio (α). The observed denaturation process could not be described by a simple reaction mechanism. It was not a process involving 2 standard reversible states, but a multi-step process. The new opportunities for investigating the kinetics process of protein denaturation can be supplied by this novel isoconversional method.

  13. Structural, Functional and Phylogenetic Analysis of Sperm Lysozyme-Like Proteins.

    PubMed

    Kalra, Shalini; Pradeep, Mangottil Ayyappan; Mohanty, Ashok K; Kaushik, Jai K

    2016-01-01

    Sperm lysozyme-like proteins belonging to c-type lysozyme family evolved in multiple forms. Lysozyme-like proteins, viz., LYZL2, LYZL3 or SLLP1, LYZL4, LYZL5 and LYZL6 are expressed in the testis of mammals. Not all members of LYZL family have been uniformly and unambiguously identified in the genome and proteome of mammals. Some studies suggested a role of SLLP1 and LYZL4 in fertilization; however, the function of other LYZL proteins is unknown. We identified all known forms of LYZL proteins in buffalo sperm by LC-MS/MS. Cloning and sequence analysis of the Lyzl cDNA showed 38-50% identity at amino acid level among the buffalo LYZL paralogs, complete conservation of eight cysteines and other signature sequences of c-type lysozyme family. Catalytic residues in SLLP1, LYZL4 and LYZL5 have undergone replacement. The substrate binding residues showed significant variation in LYZL proteins. Residues at sites 62, 101, 114 in LYZL4; 101 in SLLP1; 37, 62, and 101 in LYZL6 were more variable among diverse species. Sites 63 and 108 occupied by tryptophan were least tolerant to variation. Site 37 also showed lower tolerance to substitution in SLLP1, LYZL4 and LYZL5, but more variable in non-testicular lysozymes. Models of LYZL proteins were created by homology modeling and the substrate binding pockets were analyzed in term of binding energies and contacting residues of LYZL proteins with tri-N-acetylglucosamine (NAG)3 in the A-B-C and B-C-D binding mode. Except LYZL6, LYZL proteins did not show significant difference in binding energies in comparison to hen egg white lysozyme in the A-B-C mode. (NAG)3 binding energy in the B-C-D mode was higher by 1.3-2.2 kcal/mol than in A-B-C mode. Structural analysis indicated that (NAG)3 was involved in making more extensive interactions including hydrogen bonding with LYZL proteins in B-C-D mode than in A-B-C mode. Despite large sequence divergence among themselves and with respect to c-type lysozymes, substrate binding residues as

  14. Structural, Functional and Phylogenetic Analysis of Sperm Lysozyme-Like Proteins

    PubMed Central

    Kalra, Shalini; Pradeep, Mangottil Ayyappan; Mohanty, Ashok K.; Kaushik, Jai K.

    2016-01-01

    Sperm lysozyme-like proteins belonging to c-type lysozyme family evolved in multiple forms. Lysozyme-like proteins, viz., LYZL2, LYZL3 or SLLP1, LYZL4, LYZL5 and LYZL6 are expressed in the testis of mammals. Not all members of LYZL family have been uniformly and unambiguously identified in the genome and proteome of mammals. Some studies suggested a role of SLLP1 and LYZL4 in fertilization; however, the function of other LYZL proteins is unknown. We identified all known forms of LYZL proteins in buffalo sperm by LC-MS/MS. Cloning and sequence analysis of the Lyzl cDNA showed 38–50% identity at amino acid level among the buffalo LYZL paralogs, complete conservation of eight cysteines and other signature sequences of c-type lysozyme family. Catalytic residues in SLLP1, LYZL4 and LYZL5 have undergone replacement. The substrate binding residues showed significant variation in LYZL proteins. Residues at sites 62, 101, 114 in LYZL4; 101 in SLLP1; 37, 62, and 101 in LYZL6 were more variable among diverse species. Sites 63 and 108 occupied by tryptophan were least tolerant to variation. Site 37 also showed lower tolerance to substitution in SLLP1, LYZL4 and LYZL5, but more variable in non-testicular lysozymes. Models of LYZL proteins were created by homology modeling and the substrate binding pockets were analyzed in term of binding energies and contacting residues of LYZL proteins with tri-N-acetylglucosamine (NAG)3 in the A-B-C and B-C-D binding mode. Except LYZL6, LYZL proteins did not show significant difference in binding energies in comparison to hen egg white lysozyme in the A-B-C mode. (NAG)3 binding energy in the B-C-D mode was higher by 1.3–2.2 kcal/mol than in A-B-C mode. Structural analysis indicated that (NAG)3 was involved in making more extensive interactions including hydrogen bonding with LYZL proteins in B-C-D mode than in A-B-C mode. Despite large sequence divergence among themselves and with respect to c-type lysozymes, substrate binding residues as

  15. Insect immunity. Attacins, a family of antibacterial proteins from Hyalophora cecropia.

    PubMed

    Hultmark, D; Engström, A; Andersson, K; Steiner, H; Bennich, H; Boman, H G

    1983-01-01

    Six closely related antibacterial proteins, attacins A-F, were isolated from the hemolymph of immunized pupae of the Cecropia moth, Hyalophora cecropia. Chromatofocusing separated attacins A-F, with isoelectric points between 5.7 and 8.3. Immunological experiments show that the attacins constitute antibacterially active forms of the previously isolated inducible immune protein P5. Their mol. wts., 20-23 K, are similar to that of protein P5, but significantly lower than 28 K found for preP5 synthesized in vitro (see accompanying paper). The six attacins can be divided into two groups according to their amino acid composition and amino-terminal sequences, attacins A-D constitute a basic group and attacins E and F an acidic one. Within each group the forms are very similar. The attacins efficiently killed Escherichia coli and two other Gram-negative bacteria isolated from the gut of a silk worm but they did not act on other Gram-positive and Gram-negative bacteria tested. Only growing cells of E. coli were attacked; cells suspended in phosphate buffer were inert. Besides the cecropins and lysozyme, the attacins represent a third class of antibacterial proteins in the humoral immune system of H. cecropia.

  16. Proteomic analysis of egg white heparin-binding proteins: towards the identification of natural antibacterial molecules

    PubMed Central

    Guyot, Nicolas; Labas, Valérie; Harichaux, Grégoire; Chessé, Magali; Poirier, Jean-Claude; Nys, Yves; Réhault-Godbert, Sophie

    2016-01-01

    The chicken egg resists most environmental microbes suggesting that it potentially contains efficient antimicrobial molecules. Considering that some heparin-binding proteins in mammals are antibacterial, we investigated the presence and the antimicrobial activity of heparin-binding proteins from chicken egg white. Mass spectrometry analysis of the proteins recovered after heparin-affinity chromatography, revealed 20 proteins, including known antimicrobial proteins (avidin, lysozyme, TENP, ovalbumin-related protein X and avian bêta-defensin 11). The antibacterial activity of three new egg candidates (vitelline membrane outer layer protein 1, beta-microseminoprotein-like (LOC101750704) and pleiotrophin) was demonstrated against Listeria monocytogenes and/or Salmonella enterica Enteritidis. We showed that all these molecules share the property to inhibit bacterial growth through their heparin-binding domains. However, vitelline membrane outer layer 1 has additional specific structural features that can contribute to its antimicrobial potential. Moreover, we identified potential supplementary effectors of innate immunity including mucin 5B, E-selectin ligand 1, whey acidic protein 3, peptidyl prolyl isomerase B and retinoic acid receptor responder protein 2. These data support the concept of using heparin affinity combined to mass spectrometry to obtain an overview of the various effectors of innate immunity composing biological milieus, and to identify novel antimicrobial candidates of interest in the race for alternatives to antibiotics. PMID:27294500

  17. Hydration Potential of Lysozyme: Protein Dehydration Using a Single Microparticle Technique

    PubMed Central

    Rickard, Deborah L.; Duncan, P. Brent; Needham, David

    2010-01-01

    For biological molecules in aqueous solution, the hydration pressure as a function of distance from the molecular surface represents a very short-range repulsive pressure that limits atom-atom contact, opposing the attractive van der Waals pressure. Whereas the separation distance for molecules that easily arrange into ordered arrays (e.g., lipids, DNA, collagen fibers) can be determined from x-ray diffraction, many globular proteins are not as easily structured. Using a new micropipette technique, spherical, glassified protein microbeads can be made that allow determination of protein hydration as a function of the water activity (aw) in a surrounding medium (decanol). By adjusting aw of the dehydration medium, the final protein concentration of the solid microbead is controlled, and ranges from 700 to 1150 mg/mL. By controlling aw (and thus the osmotic pressure) around lysozyme, the repulsive pressure was determined as a function of distance between each globular, ellipsoid protein. For separation distances, d, between 2.5 and 9 Å, the repulsive decay length was 1.7 Å and the pressure extrapolated to d = 0 was 2.2 × 108 N/m2, indicating that the hydration pressure for lysozyme is similar to other biological interfaces such as phospholipid bilayers. PMID:20303865

  18. Superparamagnetic lysozyme surface-imprinted polymer prepared by atom transfer radical polymerization and its application for protein separation.

    PubMed

    Gai, Qing-Qing; Qu, Feng; Liu, Zong-Jian; Dai, Rong-Ji; Zhang, Yu-Kui

    2010-07-30

    Molecular imprinting as a promising and facile separation technique has received much attention because of their high selectivity for target molecules. In this study, the superparamagnetic lysozyme surface-imprinted polymer was prepared by a novel fabricating protocol, the grafting of the imprinted polymer on magnetic particles in aqueous media was done by atom transfer radical polymerization (ATRP), and the properties of the imprinted polymer were characterized in detail. Its high selective adsorption and recognition to lysozyme demonstrated the separation ability of the magnetic imprinted material to template molecule, and it has been used for quick and direct separation of lysozyme from the mixture of standard proteins and real egg white samples under an external magnetic field. Furthermore, the elution of lysozyme from the imprinted material was achieved by PEG/sulphate aqueous two-phase system, which caused lysozyme not only desorption from the imprinted materials but also redistribution in the top and bottom phase of aqueous two-phase system. The aqueous two-phase system exhibited some of the extraction and enrichment effect to desorbed lysozyme. Our results showed that ATRP is a promising method for the protein molecularly imprinted polymer preparation.

  19. Investigation of antibacterial mechanism and identification of bacterial protein targets mediated by antibacterial medicinal plant extracts.

    PubMed

    Yong, Ann-Li; Ooh, Keng-Fei; Ong, Hean-Chooi; Chai, Tsun-Thai; Wong, Fai-Chu

    2015-11-01

    In this paper, we investigated the antibacterial mechanism and potential therapeutic targets of three antibacterial medicinal plants. Upon treatment with the plant extracts, bacterial proteins were extracted and resolved using denaturing gel electrophoresis. Differentially-expressed bacterial proteins were excised from the gels and subjected to sequence analysis by MALDI TOF-TOF mass spectrometry. From our study, seven differentially expressed bacterial proteins (triacylglycerol lipase, N-acetylmuramoyl-L-alanine amidase, flagellin, outer membrane protein A, stringent starvation protein A, 30S ribosomal protein s1 and 60 kDa chaperonin) were identified. Additionally, scanning electron microscope study indicated morphological damages induced on bacterial cell surfaces. To the best of our knowledge, this represents the first time these bacterial proteins are being reported, following treatments with the antibacterial plant extracts. Further studies in this direction could lead to the detailed understanding of their inhibition mechanism and discovery of target-specific antibacterial agents.

  20. Hydrophobic interaction adsorption of hen egg white proteins albumin, conalbumin, and lysozyme.

    PubMed

    Rojas, Edwin E Garcia; dos Reis Coimbra, Jane S; Minim, Luis A; Saraiva, Sérgio H; da Silva, César A Sodré

    2006-08-18

    Hydrophobic adsorption equilibrium data of the hen egg white proteins albumin, conalbumin, and lysozyme were obtained in batch systems, at 25 degrees C, using the Streamline Phenyl resin as adsorbent. The influence of three types of salt, NaCl, Na(2)SO(4), or (NH(4))(2)SO(4), and their concentration on the equilibrium data were evaluated. The salt Na(2)SO(4) showed the higher interaction with the studied proteins, thus favoring the adsorption of proteins by the adsorbent, even though each type of salt interacted in a distinct manner with each protein. The isotherm models of Langmuir, Langmuir exponential, and Chen and Sun were well fitted to the equilibrium data, with no significant difference being observed at the 5% level of significance. The mass transfer model applied simulated correctly adsorption kinetics of the proteins under the studied conditions.

  1. Protein interactions in solution characterized by light and neutron scattering: comparison of lysozyme and chymotrypsinogen.

    PubMed Central

    Velev, O D; Kaler, E W; Lenhoff, A M

    1998-01-01

    The effects of pH and electrolyte concentration on protein-protein interactions in lysozyme and chymotrypsinogen solutions were investigated by static light scattering (SLS) and small-angle neutron scattering (SANS). Very good agreement between the values of the virial coefficients measured by SLS and SANS was obtained without use of adjustable parameters. At low electrolyte concentration, the virial coefficients depend strongly on pH and change from positive to negative as the pH increases. All coefficients at high salt concentration are slightly negative and depend weakly on pH. For lysozyme, the coefficients always decrease with increasing electrolyte concentration. However, for chymotrypsinogen there is a cross-over point around pH 5.2, above which the virial coefficients decrease with increasing ionic strength, indicating the presence of attractive electrostatic interactions. The data are in agreement with Derjaguin-Landau-Verwey-Overbeek (DLVO)-type modeling, accounting for the repulsive and attractive electrostatic, van der Waals, and excluded volume interactions of equivalent colloid spheres. This model, however, is unable to resolve the complex short-ranged orientational interactions. The results of protein precipitation and crystallization experiments are in qualitative correlation with the patterns of the virial coefficients and demonstrate that interaction mapping could help outline new crystallization regions. PMID:9826592

  2. pH-dependent interaction and resultant structures of silica nanoparticles and lysozyme protein.

    PubMed

    Kumar, Sugam; Aswal, Vinod K; Callow, P

    2014-02-18

    Small-angle neutron scattering (SANS) and UV-visible spectroscopy studies have been carried out to examine pH-dependent interactions and resultant structures of oppositely charged silica nanoparticles and lysozyme protein in aqueous solution. The measurements were carried out at fixed concentration (1 wt %) of three differently sized silica nanoparticles (8, 16, and 26 nm) over a wide concentration range of protein (0-10 wt %) at three different pH values (5, 7, and 9). The adsorption curve as obtained by UV-visible spectroscopy shows exponential behavior of protein adsorption on nanoparticles. The electrostatic interaction enhanced by the decrease in the pH between the nanoparticle and protein (isoelectric point ∼11.4) increases the adsorption coefficient on nanoparticles but decreases the overall amount protein adsorbed whereas the opposite behavior is observed with increasing nanoparticle size. The adsorption of protein leads to the protein-mediated aggregation of nanoparticles. These aggregates are found to be surface fractals at pH 5 and change to mass fractals with increasing pH and/or decreasing nanoparticle size. Two different concentration regimes of interaction of nanoparticles with protein have been observed: (i) unaggregated nanoparticles coexisting with aggregated nanoparticles at low protein concentrations and (ii) free protein coexisting with aggregated nanoparticles at higher protein concentrations. These concentration regimes are found to be strongly dependent on both the pH and nanoparticle size.

  3. Metal-assisted and microwave accelerated-evaporative crystallization: Application to lysozyme protein

    NASA Astrophysics Data System (ADS)

    Mauge-Lewis, Kevin

    In response to the growing need for new crystallization techniques that afford for rapid processing times along with control over crystal size and distribution, the Aslan Research Group has recently demonstrated the use of Metal-Assisted and Microwave-Accelerated Evaporative Crystallization MA-MAEC technique in conjunction with metal nanoparticles and nanostructures for the crystallization of amino acids and organic small molecules. In this study, we have employed the newly developed MA-MAEC technique to the accelerated crystallization of chicken egg-white lysozyme on circular crystallization platforms in order to demonstrate the proof-of-principle application of the method for protein crystallization. The circular crystallization platforms are constructed in-house from poly (methyl methacrylate) (PMMA) and silver nanoparticle films (SNFs), indium tin oxide (ITO) and iron nano-columns. In this study, we prove the MA-MAEC method to be a more effective technique in the rapid crystallization of macromolecules in comparison to other conventional methods. Furthermore, we demonstrate the use of the novel iCrystal system, which incorporates the use of continuous, low wattage heating to facilitate the rapid crystallization of the lysozyme while still retaining excellent crystal quality. With the incorporation of the iCrystal system, we observe crystallization times that are even shorter than those produced by the MA-MAEC technique using a conventional microwave oven in addition to significantly improved crystal quality.

  4. Time-dependent, protein-directed growth of gold nanoparticles within a single crystal of lysozyme

    SciTech Connect

    Wei, H.; Robinson, H.; Wang, Z.; Zhang, J.; House, S.; Gao, Y.-G.; Yang, L.; Tan, L. H.; Xing, H.; Hou, C.; Robertson, I. M.; Zuo, J.-M.; Lu, Y.

    2011-01-30

    Gold nanoparticles are useful in biomedical applications due to their distinct optical properties and high chemical stability. Reports of the biogenic formation of gold colloids from gold complexes has also led to an increased level of interest in the biomineralization of gold. However, the mechanism responsible for biomolecule-directed gold nanoparticle formation remains unclear due to the lack of structural information about biological systems and the fast kinetics of biomimetic chemical systems in solution. Here we show that intact single crystals of lysozyme can be used to study the time-dependent, protein-directed growth of gold nanoparticles. The protein crystals slow down the growth of the gold nanoparticles, allowing detailed kinetic studies to be carried out, and permit a three-dimensional structural characterization that would be difficult to achieve in solution. Furthermore, we show that additional chemical species can be used to fine-tune the growth rate of the gold nanoparticles.

  5. Effect of pressure on the conformation of proteins. A molecular dynamics simulation of lysozyme.

    PubMed

    McCarthy, Andrés N; Grigera, J Raúl

    2006-01-01

    The effect of pressure on the structure and mobility of lysozyme was studied by molecular dynamics computer simulation at 1 and 3 kbar (1 atm = 1.01325 bar = 101.325 kPa). The results have good agreement with the available experimental data, allowing the analysis of other features of the effect of pressure on the protein solution. The studies of mobility show that although the general mobility is restricted under pressure this is not true for some particular residues. From the analysis of secondary structure along the trajectories it is observed that the conformation under pressure is more stable, suggesting that pressure acts as a 'conformer selector' on the protein. The difference in solvent-accessed surface (SAS) with pressure shows a clear inversion of the hydrophilic/hydrophobic SAS ratio, which consequently shows that the hydrophobic interaction is considerably weaker under high hydrostatic pressure conditions.

  6. Time-dependent Protein-directed Growth of Gold Nanoparticles within a Single Crystal of Lysozyme

    SciTech Connect

    H Wei; Z Wang; J Zhang; S House; Y Gao; L Yang; H Robinson; L Tan; H Xing; C Hou

    2011-12-31

    Gold nanoparticles are useful in biomedical applications due to their distinct optical properties and high chemical stability. Reports of the biogenic formation of gold colloids from gold complexes has also led to an increased level of interest in the biomineralization of gold. However, the mechanism responsible for biomolecule-directed gold nanoparticle formation remains unclear due to the lack of structural information about biological systems and the fast kinetics of biomimetic chemical systems in solution. Here we show that intact single crystals of lysozyme can be used to study the time-dependent, protein-directed growth of gold nanoparticles. The protein crystals slow down the growth of the gold nanoparticles, allowing detailed kinetic studies to be carried out, and permit a three-dimensional structural characterization that would be difficult to achieve in solution. Furthermore, we show that additional chemical species can be used to fine-tune the growth rate of the gold nanoparticles.

  7. Lysozymes in the animal kingdom.

    PubMed

    Callewaert, Lien; Michiels, Chris W

    2010-03-01

    Lysozymes (EC 3.2.1.17) are hydrolytic enzymes, characterized by their ability to cleave the beta-(1,4)-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in peptidoglycan, the major bacterial cell wall polymer. In the animal kingdom, three major distinct lysozyme types have been identified--the c-type (chicken or conventional type), the g-type (goose-type) and the i-type (invertebrate type) lysozyme. Examination of the phylogenetic distribution of these lysozymes reveals that c-type lysozymes are predominantly present in the phylum of the Chordata and in different classes of the Arthropoda. Moreover, g-type lysozymes (or at least their corresponding genes) are found in members of the Chordata, as well as in some bivalve mollusks belonging to the invertebrates. In general, the latter animals are known to produce i-type lysozymes. Although the homology in primary structure for representatives of these three lysozyme types is limited, their three-dimensional structures show striking similarities. Nevertheless, some variation exists in their catalytic mechanisms and the genomic organization of their genes. Regarding their biological role, the widely recognized function of lysozymes is their contribution to antibacterial defence but, additionally, some lysozymes (belonging to different types) are known to function as digestive enzymes.

  8. Protein Crystal Growth Under Forced Solution Flow: Experimental Setup and General Response of Lysozyme

    NASA Technical Reports Server (NTRS)

    Vekilov, P. G.; Rosenberger, F.

    1998-01-01

    We have experimentally studied the effects of solution flow on the growth kinetics of the protein lysozyme. To this end, we have expanded our interferometry setup by a novel crystallization cell and solution recirculation system. This combination permits monitoring of interface morphology and kinetics with a depth resolution of 200 A at bulk flow rates of up to 2000 micron/s. Particular attention was paid to the prevention of protein denaturation that is often associated with the pumping of protein solutions. We found that at bulk flow rates it less than 250 microns/s the average growth rate and step velocity, R(sub avg) and upsilon(sub avg) increase with increasing it. This can be quantitatively understood in terms of the enhanced, convective solute supply to the interface. With high-purity solutions, it u greater than 250 microns/s lead to growth deceleration, and, at low supersaturations sigma, to growth cessation. When solutions containing approx. 1% of other protein impurities were used, growth deceleration occurred at any u greater than 0 and cessation in the low sigma experiments was reached at about half the it causing cessation with pure solution. The flow-induced changes in R(sub avg) and upsilon(sub avg) including growth cessation, were reversible and reproducible, independent of the direction of the u-changes and solution purity. Hence, we attribute the deceleration to the convection-enhanced supply of impurities to the interface, which at higher flow rates overpowers the effects of enhanced interfacial solute concentration. Most importantly, we found that convective transport leads to a significant reduction in kinetics fluctuations, in agreement with our earlier expectations for the lysozyme system. This supports our hypothesis that these long-term fluctuations represent an intrinsic response feature of the coupled bulk transport-interfacial kinetics system in the mixed growth control regime.

  9. The Effects of Thermal History on Nucleation of Tetragonal Lysozyme Crystals, or Hot Protein and Cold Nucleation

    NASA Technical Reports Server (NTRS)

    Burke, Michael; Judge, Russell; Pusey, Marc

    2000-01-01

    Chicken egg white lysozyme has a well characterized thermally driven phase transition. Between pH 4.2 and 5.2, the transition temperature, as defined by the point where the tetragonal and orthorhombic solubilities are equal, is a function of the pH, salt (precipitant) type and concentration, and most likely of the buffer concentration as well. This phase transition can be carried out with protein solution alone, prior to addition of precipitant solution. Warming a lysozyme solution above the phase transition point, then cooling it back below this point, has been shown to affect the subsequent nucleation rate, as determined by the numbers and size of crystals formed, but not the growth rate for the tetragonal crystal form . We have now measured the kinetics of this process and investigated its reversibility. The transition effects are progressive with temperature, having a half time of about 1 hour at 37C at pH 4.8. After holding a lysozyme solution at 37C (prior to addition of precipitant) for 16 hours, then cooling it back to 4C no return to the pre-warmed nucleation kinetics are observed after at least 4 weeks. Orthorhombic lysozyme crystals apparently do not undergo the flow-induced growth cessation of tetragonal lysozyme crystals. Putting the protein in the orthorhombic form does not affect the averaged face growth kinetics, only nucleation, for tetragonal crystals. This differential behaviour may be exploited to elucidate how and where flow affects the lysozyme crystal growth process. The presentation will focus on the results of these and ongoing studies in this area.

  10. Lysozyme Crystal

    NASA Technical Reports Server (NTRS)

    2004-01-01

    To the crystallographer, this may not be a diamond but it is just as priceless. A Lysozyme crystal grown in orbit looks great under a microscope, but the real test is X-ray crystallography. The colors are caused by polarizing filters. Proteins can form crystals generated by rows and columns of molecules that form up like soldiers on a parade ground. Shining X-rays through a crystal will produce a pattern of dots that can be decoded to reveal the arrangement of the atoms in the molecules making up the crystal. Like the troops in formation, uniformity and order are everything in X-ray crystallography. X-rays have much shorter wavelengths than visible light, so the best looking crystals under the microscope won't necessarily pass muster under the X-rays. In order to have crystals to use for X-ray diffraction studies, crystals need to be fairly large and well ordered. Scientists also need lots of crystals since exposure to air, the process of X-raying them, and other factors destroy them. Growing protein crystals in space has yielded striking results. Lysozyme's structure is well known and it has become a standard in many crystallization studies on Earth and in space.

  11. Correlation of Conformational Changes and Protein Degradation with Loss of Lysozyme Activity Due to Chlorine Dioxide Treatment.

    PubMed

    Ooi, Beng Guat; Branning, Sharon Alyssa

    2016-12-13

    Chlorine dioxide (ClO2) is a potent oxidizing agent used for the treatment of drinking water and decontamination of facilities and equipment. The purpose of this research is to elucidate the manner in which ClO2 destroys proteins by studying the effects of ClO2 on lysozyme. The degree of enzyme activity lost can be correlated to the treatment time and levels of the ClO2 used. Lysozyme activity was drastically reduced to 45.3% of original enzyme activity when exposed to 4.3 mM ClO2 in the sample after 3 h. Almost all activities were lost in 3 h after exposure to higher ClO2 concentrations of up to 16.8 and 21.9 mM. Changes in protein conformation and amount as a result of ClO2 treatment were determined using the Raman spectroscopy and gel electrophoresis. Raman shifts and the alteration of spectral features observed in the ClO2-treated lysozyme samples are associated with loss of the α-helix secondary structure, tertiary structure, and disulfide bond. Progressive degradation of the denatured lysozyme by increasing levels of chlorine dioxide was also observed in gel electrophoresis. Hence, ClO2 can effectively cause protein denaturation and degradation resulting in loss of enzyme activity.

  12. Impact of Microscale and Pilot-Scale Freeze-Drying on Protein Secondary Structures: Sucrose Formulations of Lysozyme and Catalase.

    PubMed

    Peters, Björn-Hendrik; Leskinen, Jari T T; Molnár, Ferdinand; Ketolainen, Jarkko

    2015-11-01

    Microscale (MS) freeze-drying offers rapid process cycles for early-stage formulation development. The effects of the MS approach on the secondary structures of two model proteins, lysozyme and catalase, were compared with pilot-scale (PS) vial freeze-drying. The secondary structures were assessed by attenuated total reflection Fourier transformed infrared spectroscopy. Formulations were made with increasing sucrose-protein ratios. Freeze-drying protocols involved regular cooling without thermal treatment and annealing with MS and PS equipment, and cooling rate variations with the MS. Principal component analysis of smoothed second-derivative amide I spectra revealed sucrose-protein ratio-dependent shifts toward α-helical structures. Transferability of sucrose-protein formulations from MS to PS vial freeze-drying was evidenced at regular cooling rates. Local differences in protein secondary structures between the bottom and top of sucrose-catalase samples could be detected at the sucrose-catalase ratios of 1 and 2, this being related to the initial filling height and ice crystal morphology. Annealing revealed temperature, protein, formulation, and sample location-dependent effects influencing surface morphology at the top, or causing protein secondary structure perturbation at the bottom. With the MS approach, protein secondary structure differences at different cooling rates could be detected for sucrose-lysozyme samples at the sucrose-lysozyme ratio of 1.

  13. The effect of protein-precipitant interfaces and applied shear on the nucleation and growth of lysozyme crystals.

    PubMed

    Reis, Nuno M; Chirgadze, Dimitri Y; Blundell, Tom L; Mackley, Malcolm R

    2009-11-01

    This paper is concerned with the effect of protein-precipitant interfaces and externally applied shear on the nucleation and growth kinetics of hen egg-white lysozyme crystals. The early stages of microbatch crystallization of lysozyme were explored using both optical and confocal fluorescence microscopy imaging. Initially, an antisolvent (precipitant) was added to a protein drop and the optical development of the protein-precipitant interface was followed with time. In the presence of the water-soluble polymer poly(ethylene glycol) (PEG) a sharp interface was observed to form immediately within the drop, giving an initial clear separation between the lighter protein solution and the heavier precipitant. This interface subsequently became unstable and quickly developed within a few seconds into several unstable 'fingers' that represented regions of high concentration-gradient interfaces. Confocal microscopy demonstrated that the subsequent nucleation of protein crystals occurred preferentially in the region of these interfaces. Additional experiments using an optical shearing system demonstrated that oscillatory shear significantly decreased nucleation rates whilst extending the growth period of the lysozyme crystals. The experimental observations relating to both nucleation and growth have relevance in developing efficient and reliable protocols for general crystallization procedures and the controlled crystallization of single large high-quality protein crystals for use in X-ray crystallography.

  14. Protein-salt binding data from potentiometric titrations of lysozyme in aqueous solutions containing KCl

    SciTech Connect

    Engmann, J.; Blanch, H.W.; Prausnitz, J.M. |

    1997-03-01

    An existing method for potentiometric titrations of proteins was improved, tested and applied to titrations of the enzyme hen-egg-white lysozyme in aqueous solutions containing KCl at ionic strengths from 0.1 M to 2.0 M at 25 C. Information about the protein`s net charge dependence on pH and ionic strength were obtained and salt binding numbers for the system were calculated using a linkage concept. For the pH range 2.5--11.5, the net charge slightly but distinctly increases with increasing ionic strength between 0.1 M and 2.0 M. The differences are most distinct in the pH region below 5. Above pH 11.35, the net charge decreases with increasing ionic strength. Preliminary calculation of binding numbers from titration curves at 0.1 M and 1.0 M showed selective association of chloride anions and expulsion of potassium ions at low pH. Ion-binding numbers from this work will be used to evaluate thermodynamic properties and to correlate crystallization or precipitation phase-equilibrium data in terms of a model based on the integral-equation theory of fluids which is currently under development.

  15. Biological and Clinical Implications of Lysozyme Deposition on Soft Contact Lenses.

    PubMed

    Omali, Negar Babaei; Subbaraman, Lakshman N; Coles-Brennan, Chantal; Fadli, Zohra; Jones, Lyndon W

    2015-07-01

    Within a few minutes of wear, contact lenses become rapidly coated with a variety of tear film components, including proteins, lipids, and mucins. Tears have a rich and complex composition, allowing a wide range of interactions and competitive processes, with the first event observed at the interface between a contact lens and tear fluid being protein adsorption. Protein adsorption on hydrogel contact lenses is a complex process involving a variety of factors relating to both the protein in question and the lens material. Among tear proteins, lysozyme is a major protein that has both antibacterial and anti-inflammatory functions. Contact lens materials that have high ionicity and high water content have an increased affinity to accumulate lysozyme during wear, when compared with other soft lens materials, notably silicone hydrogel lenses. This review provides an overview of tear film proteins, with a specific focus on lysozyme, and examines various factors that influence protein deposition on contact lenses. In addition, the impact of lysozyme deposition on various ocular physiological responses and bacterial adhesion to lenses and the interaction of lysozyme with other tear proteins are reviewed. This comprehensive review suggests that deposition of lysozyme on contact lens materials may provide a number of beneficial effects during contact lens wear.

  16. Protein interactions in genome maintenance as novel antibacterial targets.

    PubMed

    Marceau, Aimee H; Bernstein, Douglas A; Walsh, Brian W; Shapiro, Walker; Simmons, Lyle A; Keck, James L

    2013-01-01

    Antibacterial compounds typically act by directly inhibiting essential bacterial enzyme activities. Although this general mechanism of action has fueled traditional antibiotic discovery efforts for decades, new antibiotic development has not kept pace with the emergence of drug resistant bacterial strains. These limitations have severely restricted the therapeutic tools available for treating bacterial infections. Here we test an alternative antibacterial lead-compound identification strategy in which essential protein-protein interactions are targeted rather than enzymatic activities. Bacterial single-stranded DNA-binding proteins (SSBs) form conserved protein interaction "hubs" that are essential for recruiting many DNA replication, recombination, and repair proteins to SSB/DNA nucleoprotein substrates. Three small molecules that block SSB/protein interactions are shown to have antibacterial activity against diverse bacterial species. Consistent with a model in which the compounds target multiple SSB/protein interactions, treatment of Bacillus subtilis cultures with the compounds leads to rapid inhibition of DNA replication and recombination, and ultimately to cell death. The compounds also have unanticipated effects on protein synthesis that could be due to a previously unknown role for SSB/protein interactions in translation or to off-target effects. Our results highlight the potential of targeting protein-protein interactions, particularly those that mediate genome maintenance, as a powerful approach for identifying new antibacterial compounds.

  17. Enterococcus faecalis zinc-responsive proteins mediate bacterial defence against zinc overload, lysozyme and oxidative stress.

    PubMed

    Abrantes, Marta C; Kok, Jan; Silva Lopes, Maria de Fátima

    2014-12-01

    Two Enterococcus faecalis genes encoding the P-type ATPase EF1400 and the putative SapB protein EF0759 were previously shown to be strongly upregulated in the presence of high concentrations of zinc. In the present work, we showed that a Zn(2+)-responsive DNA-binding motif (zim) is present in the promoter regions of these genes. Both proteins were further studied with respect to their involvement in zinc homeostasis and invasion of the host. EF0759 contributed to intramacrophage survival by an as-yet unknown mechanism(s). EF1400, here renamed ZntAEf, is an ATPase with specificity for zinc and plays a role in dealing with several host defences, i.e. zinc overload, oxidative stress and lysozyme; it provides E. faecalis cells with the ability to survive inside macrophages. As these three host defence mechanisms are important at several sites in the host, i.e. inside macrophages and in saliva, this work suggested that ZntAEf constitutes a crucial E. faecalis defence mechanism that is likely to contribute to the ability of this bacterium to endure life inside its host.

  18. Glycation of Lysozyme by Glycolaldehyde Provides New Mechanistic Insights in Diabetes-Related Protein Aggregation.

    PubMed

    Mariño, Laura; Maya-Aguirre, Carlos Andrés; Pauwels, Kris; Vilanova, Bartolomé; Ortega-Castro, Joaquin; Frau, Juan; Donoso, Josefa; Adrover, Miquel

    2017-03-14

    Glycation occurs in vivo as a result of the nonenzymatic reaction of carbohydrates (and/or their autoxidation products) with proteins, DNA, or lipids. Protein glycation causes loss-of-function and, consequently, the development of diabetic-related diseases. Glycation also boosts protein aggregation, which can be directly related with the higher prevalence of aggregating diseases in diabetic people. However, the molecular mechanism connecting glycation with aggregation still remains unclear. Previously we described mechanistically how glycation of hen egg-white lysozyme (HEWL) with ribose induced its aggregation. Here we address the question of whether the ribose-induced aggregation is a general process or it depends on the chemical nature of the glycating agent. Glycation of HEWL with glycolaldehyde occurs through two different scenarios depending on the HEWL concentration regime (both within the micromolar range). At low HEWL concentration, non-cross-linking fluorescent advanced glycation end-products (AGEs) are formed on Lys side chains, which do not change the protein structure but inhibit its enzymatic activity. These AGEs have little impact on HEWL surface hydrophobicity and, therefore, a negligible effect on its aggregation propensity. Upon increasing HEWL concentration, the glycation mechanism shifts toward the formation of intermolecular cross-links, which triggers a polymerization cascade involving the formation of insoluble spherical-like aggregates. These results notably differ with the aggregation-modulation mechanism of ribosylated HEWL directed by hydrophobic interactions. Additionally, their comparison constitutes the first experimental evidence showing that the mechanism underlying the aggregation of a glycated protein depends on the chemical nature of the glycating agent.

  19. T4 phage lysozyme: a protein designed for understanding tryptophan photophysics

    NASA Astrophysics Data System (ADS)

    Hudson, Bruce S.; Harris, Dan

    1990-05-01

    Bacteriophage T4 lysozyme in its wild type form contains three tryptophan residues (at sequence postions 126, 138 and 158). These three residues are in rather different environments in the protein: 126 and 158 are near the protein surface while residue 138 is more buried. T4 lysozyme has been genetically engineered to prepare all possible variants in which one or more of the tryptophan residues have been replaced by tyrosine. The available data supports the hypothesis that this substitution has, at most, a very minor effect on the structure of the protein. The three species with single tryptophan residues have been investigated in detail. The surface location of residue 126 compared to the buried location of residue 138 is reflected in the difference in collisional quenching observed with added potassium iodide. It is found that the spectral and radiative properties of the three proteins are very similar but that their radiationless decay properties are quite distinct. This is apparently due to short-range collisional quenching by neighboring side chains. Comparison with solution quenching measurements permits the identification of the specific quenching groups involved for each tryptophan residue and provides a semi-quantitative rationale for the radiationless decay rate. This collisional quenching interpretation is supported by mutational effects on fluorescence quantum yield. This simple picture of the behavior of these single-tryptophan proteins is clearly revealed in this particular case because of the unambiguous choice of collisional quenching groups. The time dependence of the fluorescence decay of each of these single-tryptophan proteins is quite complex. Several methods of analysis are presented and discussed in terms of their underlying physical basis. Internal collisional quenching, as suggested from the comparative studies, is expected to lead to non-exponential behavior. This is consistent with the observed time dependence. Analysis of the temporal

  20. SpoVG Is a Conserved RNA-Binding Protein That Regulates Listeria monocytogenes Lysozyme Resistance, Virulence, and Swarming Motility

    PubMed Central

    Burke, Thomas P.

    2016-01-01

    ABSTRACT In this study, we sought to characterize the targets of the abundant Listeria monocytogenes noncoding RNA Rli31, which is required for L. monocytogenes lysozyme resistance and pathogenesis. Whole-genome sequencing of lysozyme-resistant suppressor strains identified loss-of-expression mutations in the promoter of spoVG, and deletion of spoVG rescued lysozyme sensitivity and attenuation in vivo of the rli31 mutant. SpoVG was demonstrated to be an RNA-binding protein that interacted with Rli31 in vitro. The relationship between Rli31 and SpoVG is multifaceted, as both the spoVG-encoded protein and the spoVG 5′-untranslated region interacted with Rli31. In addition, we observed that spoVG-deficient bacteria were nonmotile in soft agar and suppressor mutations that restored swarming motility were identified in the gene encoding a major RNase in Gram-positive bacteria, RNase J1. Collectively, these findings suggest that SpoVG is similar to global posttranscriptional regulators, a class of RNA-binding proteins that interact with noncoding RNA, regulate genes in concert with RNases, and control pleiotropic aspects of bacterial physiology. PMID:27048798

  1. Mouse SLLP1, a sperm lysozyme-like protein involved in sperm-egg binding and fertilization.

    PubMed

    Herrero, María Belén; Mandal, Arabinda; Digilio, Laura C; Coonrod, Scott A; Maier, Bernhard; Herr, John C

    2005-08-01

    This study demonstrates the retention of mouse sperm lysozyme-like protein (mSLLP1) in the equatorial segment of spermatozoa following the acrosome reaction and a role for mSLLP1 in sperm-egg binding and fertilization. Treatment of cumulus intact oocytes with either recmSLLP1 or its antiserum resulted in a significant (P < or = 0.05) inhibition of fertilization. Co-incubation of zona-free mouse oocytes with capacitated mouse spermatozoa in the presence of varying concentrations of anti-recmSLLP1 serum or recmSLLP1 also inhibited sperm-oolemma binding. A complete inhibition of binding and fusion of spermatozoa to the oocyte occurred at 12.5 muM concentration of recmSLLP1, while conventional chicken and human lysozymes did not block sperm-egg binding. mSLLP1 showed receptor sites in the perivitelline space as well as on the microvillar region of the egg plasma membrane. The retention of mSLLP1 in the equatorial segment of acrosome-reacted sperm, the inhibitory effects of both recmSLLP1 and antibodies to SLLP1 on in vitro fertilization with both cumulus intact and zona-free eggs, and the definition of complementary SLLP1-binding sites on the egg plasma membrane together support the hypothesis that a c lysozyme-like protein is involved in the binding of spermatozoa to the egg plasma membrane during fertilization.

  2. Molecular cloning, expression of orange-spotted grouper goose-type lysozyme cDNA, and lytic activity of its recombinant protein.

    PubMed

    Yin, Zhi-Xin; He, Jian-Guo; Deng, Wei-Xin; Chan, Siu-Ming

    2003-07-08

    Lysozyme acts as a non-specific innate-immunity molecule against the invasion of bacteria pathogens. A leukocyte cDNA library of orange-spotted grouper Epinephelus coioides was constructed and the goose-type (g-type) lysozyme cDNA was isolated. The complete cDNA consists of an open reading frame of 585 bp encoding a protein of 194 amino acids. This protein shows a 72.2% amino acid sequence identity with the flounder g-type lysozyme. Similar to most other species, the glu catalytic residue in g-type lysozymes of the grouper is conserved. Furthermore, like the flounder and carp, the 4 conserved cysteine residues identified in avian and mammalian g-type lysozymes were also absent from the grouper. Northern blot analysis indicated that the g-type lysozyme was expressed in intestine, liver, spleen, anterior kidney, posterior kidney, heart, gill, muscle and leukocytes. In addition, RT-PCR analysis detected the g-type lysozyme transcripts in the stomach, brain and ovary. When an orange-spotted grouper was injected with Vibrio alginolyticus, the number of lysozyme mRNA transcripts detected in the stomach, spleen, anterior kidney, posterior kidney, heart, brain and leucocytes increased 72 h after injection. Recombinant grouper g-type lysozyme produced in the Escherichia coli expression system showed lytic activity against Micrococcus lysodeikticus, V. alginolyticus from Epinephelus fario, V. vulnificus from culture water, Aeromonas hydrophila from soft-shell turtle, A. hydrophila from goldfish and V. parahaemolyticus, Pseudomonas fluorescens and V. fluvialis from culture water.

  3. Generation and properties of antibacterial coatings based on electrostatic attachment of silver nanoparticles to protein-coated polypropylene fibers.

    PubMed

    Goli, Kiran K; Gera, Nimish; Liu, Xiaomeng; Rao, Balaji M; Rojas, Orlando J; Genzer, Jan

    2013-06-12

    We present a simple method for attaching silver nanoparticles to polypropylene (PP) fibers in a two-step process to impart antibacterial properties. Specifically, PP fibers are pretreated by the adsorption from an aqueous solution of heat-denatured lysozyme (LYS) followed by LYS cross-linking using glutaraldehyde and sodium borohydride. At neutral pH, the surface of the adsorbed LYS layer is enriched with numerous positive charges. Silver nanoparticles (AgNPs) capped with trisodium citrate are subsequently deposited onto the protein-coated PP. Nanoparticle binding is mediated by electrostatic interactions between the positively charged LYS layer and the negatively charged AgNPs. The density of AgNPs deposited on PP depends on the amount of protein adsorbed on the surface. UV-vis spectroscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, and scanning electron microscopy are employed to follow all preparation steps and to characterize the resulting functional surfaces. The antibacterial activity of the modified surfaces is tested against gram negative bacteria Escherichia coli (E. coli). Overall, our results show that PP surfaces coated with AgNPs exhibit excellent antibacterial activity with 100% removal efficiency.

  4. Antibacterial discovery in actinomycetes strains with mutations in RNA polymerase or ribosomal protein S12.

    PubMed

    Hosaka, Takeshi; Ohnishi-Kameyama, Mayumi; Muramatsu, Hideyuki; Murakami, Kana; Tsurumi, Yasuhisa; Kodani, Shinya; Yoshida, Mitsuru; Fujie, Akihiko; Ochi, Kozo

    2009-05-01

    We show that selection of drug-resistant bacterial mutants allows the discovery of antibacterial compounds. Mutant strains of a soil-isolated Streptomyces species that does not produce antibacterials synthesize a previously unknown class of antibacterial, which we name piperidamycin. Overall, 6% of non-Streptomyces actinomycetes species and 43% of Streptomyces species that do not produce antibacterials are activated to produce them. The antibacterial-producing mutants all carried mutations in RNA polymerase and/or the ribosomal protein S12.

  5. Characteristics of hydration water around hen egg lysozyme as the protein model in aqueous solution. FTIR spectroscopy and molecular dynamics simulation.

    PubMed

    Panuszko, Aneta; Wojciechowski, Marek; Bruździak, Piotr; Rakowska, Paulina W; Stangret, Janusz

    2012-12-05

    In this paper, the hydration of a model protein--hen egg white lysozyme in aqueous solution has been presented. The leading method used was FTIR spectroscopy with an application of a technique of semi-heavy water (HDO) isotope dilution. Analysis of spectra of HDO isotopically diluted in water solution of lysozyme allowed us to isolate HDO spectra affected by lysozyme, and thus to characterise the energetic state of water molecules and their arrangement around protein molecules. The number of water molecules and the shape of the affected HDO spectrum were obtained using a classical and a chemometric method. This shape showed that the HDO spectrum affected by lysozyme may be presented as a superposition of two spectra corresponding to HDO affected by N-methylacetamide and the carboxylate anion (of the formic acid). Moreover, based on the difference in intermolecular distances distribution of water molecules (obtained from spectral data), we demonstrated that the lysozyme molecule causes a decrease in population of weak hydrogen bonds, and concurrently increases the probability of an occurrence of short hydrogen bonds in water affected by lysozyme. This conclusion was also confirmed by the molecular dynamics (MD) simulation.

  6. The lysozyme from insect (Manduca sexta) is a cold-adapted enzyme

    PubMed Central

    Sotelo-Mundo, Rogerio R.; Lopez-Zavala, Alonso A.; Garcia-Orozco, Karina D.; Arvizu-Flores, Aldo A.; Velazquez-Contreras, Enrique F.; Valenzuela-Soto, Elisa M.; Rojo-Dominguez, Arturo; Kanost, Michael R.

    2008-01-01

    Enzymatic activity is dependent on temperature, although some proteins have evolved to retain activity at low temperatures at the expense of stability. Cold adapted enzymes are present in a variety of organisms and there is ample interest in their structure-function relationships. Lysozyme (E.C. 3.2.1.17) is one of the most studied enzymes due to its antibacterial activity against Gram positive bacteria and is also a cold adapted protein. In this work the characterization of lysozyme from the insect Manduca sexta and its activity at low temperatures is presented. Both M. sexta lysozymes natural and recombinant showed a higher content of α-helix secondary structure compared to that of hen egg white lysozyme and a higher specific enzymatic activity in the range of 5−30 °C. These results together with measured thermodynamical activation parameters support the designation of M. sexta lysozyme as a cold adapted enzyme. Therefore, the insect recombinant lysozyme is feasible as a model for structure-function studies for cold-adapted proteins. PMID:17979817

  7. The Lysozyme from Insect (Manduca sexta) is a Cold-Adapted Enzyme

    SciTech Connect

    Sotelo-Mundo,R.; Lopez-Zavala, A.; Garcia-Orozco, K.; Arvizu-Flores, A.; Velazquez-Contreras, E.; Valenzuela-Soto, E.; Rojo-Dominguez, A.; Kanost, M.

    2007-01-01

    Enzymatic activity is dependent on temperature, although some proteins have evolved to retain activity at low temperatures at the expense of stability. Cold adapted enzymes are present in a variety of organisms and there is ample interest in their structure-function relationships. Lysozyme (E.C. 3.2.1.17) is one of the most studied enzymes due to its antibacterial activity against Gram positive bacteria and is also a cold adapted protein. In this work the characterization of lysozyme from the insect Manduca sexta and its activity at low temperatures is presented. Both M. sexta lysozymes natural and recombinant showed a higher content of {alpha}-helix secondary structure compared to that of hen egg white lysozyme and a higher specific enzymatic activity in the range of 5-30 {sup o}C. These results together with measured thermodynamic activation parameters support the designation of M. sexta lysozyme as a cold adapted enzyme. Therefore, the insect recombinant lysozyme is feasible as a model for structure-function studies for cold-adapted proteins.

  8. Stokes-flow computation of the diffusion coefficient and rotational diffusion tensor of lysozyme, a globular protein

    NASA Astrophysics Data System (ADS)

    Zhao, Hong; Pearlstein, Arne J.

    2002-07-01

    Based on a closed surface of triangles fitted to atomic coordinates determined crystallographically, Brune and Kim [Proc. Natl. Acad. Sci. USA 90, 3835-3839 (1993)] proposed a boundary-element Stokes-flow technique for ab initio computation of a translational diffusion coefficient and the rotational diffusion tensor Dr of globular proteins. They applied their approach to atomic coordinates for a tetragonal structure of hen egg-white lysozyme, and reported that computed values of a translational diffusion coefficient and Dr=tr(Dr)/3 agreed well with experiment. After establishing the identity between the infinite-dilution tracer diffusion coefficient of the protein macroion (D+ for lysozyme cation) and the "translational diffusion coefficient" computed by Brune and Kim, we adopt a somewhat different computational approach and show how convergence of D+ and Dr for tetragonal lysozyme depends on two computational parameters characterizing the fidelity of the geometric approximation to the protein surface and two others characterizing the accuracy of the Stokes-flow computations. We then compute D+ and Dr for lysozyme using atomic coordinates for the triclinic crystal structure, three structures determined by nuclear magnetic resonance spectroscopy in the liquid phase (presumably corresponding more closely to in vivo structures), the solvated tetragonal structure (with 108 water molecules) considered by Brune and Kim, and a "dry" version of the same structure. These computations show that D+ and Dr computed for all of the dry crystal structures are in excellent agreement with those for the liquid-phase conformations. Values of D+ and Dr computed for the solvated structure are lower, consistent with the larger volume and area of the corresponding polyhedral surface. We also show that several choices of the origin of the force system [discussed by Brenner, J. Colloid Interface Sci. 23, 407-436 (1967)] give rise to nearly identical translational diffusion coefficients

  9. Molecular cloning, sequence analysis and phylogeny of first caudata g-type lysozyme in axolotl (Ambystoma mexicanum).

    PubMed

    Yu, Haining; Gao, Jiuxiang; Lu, Yiling; Guang, Huijuan; Cai, Shasha; Zhang, Songyan; Wang, Yipeng

    2013-11-01

    Lysozymes are key proteins that play important roles in innate immune defense in many animal phyla by breaking down the bacterial cell-walls. In this study, we report the molecular cloning, sequence analysis and phylogeny of the first caudate amphibian g-lysozyme: a full-length spleen cDNA library from axolotl (Ambystoma mexicanum). A goose-type (g-lysozyme) EST was identified and the full-length cDNA was obtained using RACE-PCR. The axolotl g-lysozyme sequence represents an open reading frame for a putative signal peptide and the mature protein composed of 184 amino acids. The calculated molecular mass and the theoretical isoelectric point (pl) of this mature protein are 21523.0 Da and 4.37, respectively. Expression of g-lysozyme mRNA is predominantly found in skin, with lower levels in spleen, liver, muscle, and lung. Phylogenetic analysis revealed that caudate amphibian g-lysozyme had distinct evolution pattern for being juxtaposed with not only anura amphibian, but also with the fish, bird and mammal. Although the first complete cDNA sequence for caudate amphibian g-lysozyme is reported in the present study, clones encoding axolotl's other functional immune molecules in the full-length cDNA library will have to be further sequenced to gain insight into the fundamental aspects of antibacterial mechanisms in caudate.

  10. Role of the solvent in the dynamical transitions of proteins: the case of the lysozyme-water system.

    PubMed

    Mallamace, Francesco; Chen, Sow-Hsin; Broccio, Matteo; Corsaro, Carmelo; Crupi, Vincenza; Majolino, Domenico; Venuti, Valentina; Baglioni, Piero; Fratini, Emiliano; Vannucci, Chiara; Stanley, H Eugene

    2007-07-28

    We study the dynamics of hydration water in the protein lysozyme in the temperature range 180 Kprotein hydration water. Below the first transition, at about 220 K, the hydration water displays an unambiguous fragile-to-strong dynamic crossover, resulting in the loss of the protein conformational flexibility. Above the second transition, at about 346 K, where the protein unfolds, the dynamics of the hydration water appears to be dominated by the non-hydrogen-bonded fraction of water molecules.

  11. Role of the solvent in the dynamical transitions of proteins: The case of the lysozyme-water system

    NASA Astrophysics Data System (ADS)

    Mallamace, Francesco; Chen, Sow-Hsin; Broccio, Matteo; Corsaro, Carmelo; Crupi, Vincenza; Majolino, Domenico; Venuti, Valentina; Baglioni, Piero; Fratini, Emiliano; Vannucci, Chiara; Stanley, H. Eugene

    2007-07-01

    We study the dynamics of hydration water in the protein lysozyme in the temperature range 180Kprotein hydration water. Below the first transition, at about 220K, the hydration water displays an unambiguous fragile-to-strong dynamic crossover, resulting in the loss of the protein conformational flexibility. Above the second transition, at about 346K, where the protein unfolds, the dynamics of the hydration water appears to be dominated by the non-hydrogen-bonded fraction of water molecules.

  12. New sub-family of lysozyme-like proteins shows no catalytic activity: crystallographic and biochemical study of STM3605 protein from Salmonella Typhimurium

    SciTech Connect

    Michalska, Karolina; Brown, Roslyn N.; Li, Hui; Jedrzejczak, Robert; Niemann, George; Heffron, Fred; Cort, John R.; Adkins, Joshua N.; Babnigg, Gyorgy; Joachimiak, Andrzej

    2013-03-01

    Phage viruses that infect prokaryotes integrate their genome into the host chromosome; thus, microbial genomes typically contain genetic remnants of both recent and ancient phage infections. Often phage genes occur in clusters of atypical G+C content that reflect integration of the foreign DNA. However, some phage genes occur in isolation without other phage gene neighbors, probably resulting from horizontal gene transfer. In these cases, the phage gene product is unlikely to function as a component of a mature phage particle, and instead may have been co-opted by the host for its own benefit. The product of one such gene from Salmonella enterica serovar Typhimurium, STM3605, encodes a protein with modest sequence similarity to phage-like lysozyme (N-acetylmuramidase) but appears to lack essential catalytic residues that are strictly conserved in all lysozymes. Close homologs in other bacteria share this characteristic. The structure of the STM3605 protein was characterized by X-ray crystallography, and functional assays showed that it is a stable, folded protein whose structure closely resembles lysozyme. However, this protein is unlikely to hydrolyze peptidoglycan. Instead, STM3605 is presumed to have evolved an alternative function because it shows some lytic activity and partitions to micelles.

  13. Molecular Characterization of a Lysozyme Gene and Its Altered Expression Profile in Crowded Beet Webworm (Loxostege sticticalis)

    PubMed Central

    Kong, Hailong; Lv, Min; Mao, Nian; Wang, Cheng; Cheng, Yunxia; Zhang, Lei; Jiang, Xingfu; Luo, Lizhi

    2016-01-01

    There is growing evidence that insects living in high-density populations exhibit an increase in immune function to counter a higher risk of disease. This phenomenon, known as density-dependent prophylaxis, has been experimentally tested in a number of insect species. Although density-dependent prophylaxis is especially prevalent in insects exhibiting density-dependent phase polyphenism, the molecular mechanism remains unclear. Our previous study demonstrated that the antibacterial activity of lysozyme is important for this process in the beet webworm Loxostege sticticalis. In this study, a lysozyme cDNA from L. sticticalis was cloned and characterized. The full-length cDNA is 1078 bp long and contains an open reading frame of 426 bp that encodes 142 amino acids. The deduced protein possesses structural characteristics of a typical c-type lysozyme and clusters with c-type lysozymes from other Lepidoptera. LsLysozyme was found to be expressed throughout all developmental stages, showing the highest level in pupae. LsLysozyme was also highly expressed in the midgut and fat body. Elevated LsLysozyme expression was observed in L. sticticalis larvae infected by Beauveria bassiana and in larvae reared under crowding conditions. In addition, the expression level of LsLysozyme in infected larvae reared at a density of 10 larvae per jar was significantly higher compared to those reared at a density of l or 30 larvae per jar. These results suggest that larval crowding affects the gene expression profile of this lysozyme. This study provides additional insight into the expression of an immune-associated lysozyme gene and helps us to better understand the immune response of L. sticticalis under crowding conditions. PMID:27575006

  14. High-pressure protein crystallography of hen egg-white lysozyme

    SciTech Connect

    Yamada, Hiroyuki; Nagae, Takayuki; Watanabe, Nobuhisa

    2015-04-01

    The crystal structure of hen egg-white lysozyme (HEWL) was analyzed under pressures of up to 950 MPa. The high pressure modified the conformation of the molecule and induced a novel phase transition in the tetragonal crystal of HEWL. Crystal structures of hen egg-white lysozyme (HEWL) determined under pressures ranging from ambient pressure to 950 MPa are presented. From 0.1 to 710 MPa, the molecular and internal cavity volumes are monotonically compressed. However, from 710 to 890 MPa the internal cavity volume remains almost constant. Moreover, as the pressure increases to 950 MPa, the tetragonal crystal of HEWL undergoes a phase transition from P4{sub 3}2{sub 1}2 to P4{sub 3}. Under high pressure, the crystal structure of the enzyme undergoes several local and global changes accompanied by changes in hydration structure. For example, water molecules penetrate into an internal cavity neighbouring the active site and induce an alternate conformation of one of the catalytic residues, Glu35. These phenomena have not been detected by conventional X-ray crystal structure analysis and might play an important role in the catalytic activity of HEWL.

  15. Hen egg-white lysozyme crystallisation: protein stacking and structure stability enhanced by a Tellurium(VI)-centred polyoxotungstate.

    PubMed

    Bijelic, Aleksandar; Molitor, Christian; Mauracher, Stephan G; Al-Oweini, Rami; Kortz, Ulrich; Rompel, Annette

    2015-01-19

    As synchrotron radiation becomes more intense, detectors become faster and structure-solving software becomes more elaborate, obtaining single crystals suitable for data collection is now the bottleneck in macromolecular crystallography. Hence, there is a need for novel and advanced crystallisation agents with the ability to crystallise proteins that are otherwise challenging. Here, an Anderson-Evans-type polyoxometalate (POM), specifically Na6 [TeW6 O24 ]⋅22 H2 O (TEW), is employed as a crystallisation additive. Its effects on protein crystallisation are demonstrated with hen egg-white lysozyme (HEWL), which co-crystallises with TEW in the vicinity (or within) the liquid-liquid phase separation (LLPS) region. The X-ray structure (PDB ID: 4PHI) determination revealed that TEW molecules are part of the crystal lattice, thus demonstrating specific binding to HEWL with electrostatic interactions and hydrogen bonds. The negatively charged TEW polyoxotungstate binds to sites with a positive electrostatic potential located between two (or more) symmetry-related protein chains. Thus, TEW facilitates the formation of protein-protein interfaces of otherwise repulsive surfaces, and thereby the realisation of a stable crystal lattice. In addition to retaining the isomorphicity of the protein structure, the anomalous scattering of the POMs was used for macromolecular phasing. The results suggest that hexatungstotellurate(VI) has great potential as a crystallisation additive to promote both protein crystallisation and structure elucidation.

  16. Hen Egg-White Lysozyme Crystallisation: Protein Stacking and Structure Stability Enhanced by a Tellurium(VI)-Centred Polyoxotungstate

    PubMed Central

    Bijelic, Aleksandar; Molitor, Christian; Mauracher, Stephan G; Al-Oweini, Rami; Kortz, Ulrich; Rompel, Annette

    2015-01-01

    As synchrotron radiation becomes more intense, detectors become faster and structure-solving software becomes more elaborate, obtaining single crystals suitable for data collection is now the bottleneck in macromolecular crystallography. Hence, there is a need for novel and advanced crystallisation agents with the ability to crystallise proteins that are otherwise challenging. Here, an Anderson–Evans-type polyoxometalate (POM), specifically Na6[TeW6O24]⋅22 H2O (TEW), is employed as a crystallisation additive. Its effects on protein crystallisation are demonstrated with hen egg-white lysozyme (HEWL), which co-crystallises with TEW in the vicinity (or within) the liquid–liquid phase separation (LLPS) region. The X-ray structure (PDB ID: 4PHI) determination revealed that TEW molecules are part of the crystal lattice, thus demonstrating specific binding to HEWL with electrostatic interactions and hydrogen bonds. The negatively charged TEW polyoxotungstate binds to sites with a positive electrostatic potential located between two (or more) symmetry-related protein chains. Thus, TEW facilitates the formation of protein–protein interfaces of otherwise repulsive surfaces, and thereby the realisation of a stable crystal lattice. In addition to retaining the isomorphicity of the protein structure, the anomalous scattering of the POMs was used for macromolecular phasing. The results suggest that hexatungstotellurate(VI) has great potential as a crystallisation additive to promote both protein crystallisation and structure elucidation. PMID:25521080

  17. High-pressure protein crystallography of hen egg-white lysozyme

    PubMed Central

    Yamada, Hiroyuki; Nagae, Takayuki; Watanabe, Nobuhisa

    2015-01-01

    Crystal structures of hen egg-white lysozyme (HEWL) determined under pressures ranging from ambient pressure to 950 MPa are presented. From 0.1 to 710 MPa, the molecular and internal cavity volumes are monotonically compressed. However, from 710 to 890 MPa the internal cavity volume remains almost constant. Moreover, as the pressure increases to 950 MPa, the tetragonal crystal of HEWL undergoes a phase transition from P43212 to P43. Under high pressure, the crystal structure of the enzyme undergoes several local and global changes accompanied by changes in hydration structure. For example, water molecules penetrate into an internal cavity neighbouring the active site and induce an alternate conformation of one of the catalytic residues, Glu35. These phenomena have not been detected by conventional X-ray crystal structure analysis and might play an important role in the catalytic activity of HEWL. PMID:25849385

  18. A method for rapid liquid-solid phase solubility measurements using the protein lysozyme

    NASA Technical Reports Server (NTRS)

    Pusey, Marc L.; Gernert, Kim

    1988-01-01

    Using hen's egg white lysozyme crystals as the test material, a simple system was developed for rapidly and unambiguously determining solubilities in (aqueous) solutions. The system is based upon a maximization of the solid surface area available for solute transfer to or from the solution, and a minimization of both the solution volume which must be equilibrated and the distance over which diffusive solute exchange occurs. This technique is further enhanced by using duplicate test systems which differ only in that one approaches equilibrium from an oversaturated solution, while the other from an undersaturated solution. Thus, the resulting data pair brackets the solubility value. In practical terms, the data points are found to usually be within 3 percent of each other, and individual solubility data points may usually be made at this resolution within 8-24 h depending upon the temperature change made since the previous determination.

  19. Functionalized nanosponges for controlled antibacterial and antihypocalcemic actions.

    PubMed

    Deshmukh, Kiran; Tanwar, Yuveraj Singh; Sharma, Shailendra; Shende, Pravin; Cavalli, Roberta

    2016-12-01

    The aim of the present work was to develop lysozyme impregnated surface-active nanosponges to maintain its conformational stability and break bacterial cell walls by catalyzing the hydrolysis of 1,4-β-linkages between N-acetyl-d-glucosamine and N-acetylmuramic acid residues present in peptidoglycan layer surrounding the bacterial cell membrane, and for controlling the release of calcium in hypocalcemia condition. Different carbonyl diimidazole cross-linked β-cyclodextrin nanosponges with and without CaCO3 and CMC were prepared by polymer condensation method. The surface-active nanosponges were impregnated by lysozyme due to their ability to adsorb protein. Lysozyme impregnated nanosponges had a monomodal particle size distribution of 347.46±3.07 to 550.34±5.23nm, with a narrow distribution. The zeta potentials were sufficiently increased upon lysozyme impregnation, suggesting stable formulations by preventing aggregation. The in vitro release studies showed controlled release of lysozyme and calcium over a period of 24h. FTIR studies confirmed the impregnation of lysozyme on nanosponges and encapsulation of calcium in nanosponges. Lysozyme formulation showed promising conformational stability by DSC. It can be concluded that the stable nanosponges formulation is a promising carrier for antibacterial protein and preventing depletion of calcium in antibiotic associated hypocalcemic condition.

  20. Electrostatic model for protein adsorption in ion-exchange chromatography and application to monoclonal antibodies, lysozyme and chymotrypsinogen A.

    PubMed

    Guélat, Bertrand; Ströhlein, Guido; Lattuada, Marco; Morbidelli, Massimo

    2010-08-27

    A model for the adsorption equilibrium of proteins in ion-exchange chromatography explicitly accounting for the effect of pH and salt concentration in the limit of highly diluted systems was developed. It is based on the use of DLVO theory to estimate the electrostatic interactions between the charged surface of the ion-exchanger and the proteins. The corresponding charge distributions were evaluated as a function of pH and salt concentration using a molecular approach. The model was verified for the adsorption equilibrium of lysozyme, chymotrypsinogen A and four industrial monoclonal antibodies on two strong cation-exchangers. The adsorption equilibrium constants of these proteins were determined experimentally at various pH values and salt concentrations and the model was fitted with a good agreement using three adjustable parameters for each protein in the whole range of experimental conditions. Despite the simplifications of the model regarding the geometry of the protein-ion-exchanger system, the physical meaning of the parameters was retained.

  1. Electrolyte effect on the phase behavior of silica nanoparticles with lysozyme and bovine-serum-albumin proteins

    NASA Astrophysics Data System (ADS)

    Yadav, Indresh; Aswal, V. K.; Kohlbrecher, J.

    2015-05-01

    Small-angle neutron scattering (SANS) and dynamic light scattering (DLS) studies have been carried out to investigate the effect of an electrolyte on the phase behavior of anionic silica nanoparticles with two globular proteins—cationic lysozyme [molecular weight (MW) 14.7 kDa] and anionic bovine serum albumin (MW 66.4 kDa). The results are compared with our earlier published work on similar systems without any electrolyte [I. Yadav, S. Kumar, V. K. Aswal, and J. Kohlbrecher, Phys. Rev. E 89, 032304 (2014), 10.1103/PhysRevE.89.032304]. Both the nanoparticle-protein systems transform to two phase at lower concentration of protein in the presence of an electrolyte. The autocorrelation function in DLS suggests that the diffusion coefficient (D) of a nanoparticle-protein system decreases in approaching two phase with the increase in protein concentration. This variation in D can be attributed to increase in attractive interaction and/or overall increase in the size. Further, these two contributions (interaction and structure) are determined from the SANS data. The changes in the phase behavior of nanoparticle-protein systems in the presence of an electrolyte are explained in terms of modifications in both the repulsive and attractive components of interaction between nanoparticles. In a two-phase system individual silica nanoparticles coexist along with their fractal aggregates.

  2. Hydrophobic clustering in nonnative states of a protein: Interpretation of chemical shifts in NMR spectra of denatured states of lysozyme

    SciTech Connect

    Evans, P.A.; Topping, K.D.; Woolfson, D.N.; Dobson, C.M. )

    1991-01-01

    Chemical shifts of resonances of specific protons in the 1H NMR spectrum of thermally denatured hen lysozyme have been determined by exchange correlation with assigned native state resonances in 2D NOESY spectra obtained under conditions where the two states are interconverting. There are subtle but widespread deviations of the measured shifts from the values which would be anticipated for a random coil; in the case of side chain protons these are virtually all net upfield shifts and it is shown that this may be the averaged effect of interactions with aromatic rings in a partially collapsed denatured state. In a very few cases, notably that of two sequential tryptophan residues, it is possible to interpret these effects in terms of specific, local interresidue interactions. Generally, however, there is no correlation with either native state shift perturbations or with sequence proximity to aromatic groups. Diminution of most of the residual shift perturbations on reduction of the disulfide cross-links confirms that they are not simply effects of residues adjacent in the sequence. Similar effects of chemical denaturants, with the disulfides intact, demonstrate that the shift perturbations reflect an enhanced tendency to side chain clustering in the thermally denatured state. The temperature dependences of the shift perturbations suggest that this clustering is noncooperative and is driven by small, favorable enthalpy changes. While the extent of conformational averaging is clearly much greater than that observed for a homologous protein, alpha-lactalbumin, in its partially folded molten globule state, the results clearly show that thermally denatured lysozyme differs substantially from a random coil, principally in that it is partially hydrophobically collapsed.

  3. Titanium Surface Priming with Phase-Transited Lysozyme to Establish a Silver Nanoparticle-Loaded Chitosan/Hyaluronic Acid Antibacterial Multilayer via Layer-by-Layer Self-Assembly

    PubMed Central

    Zhong, Xue; Song, Yunjia; Yang, Peng; Wang, Yao; Jiang, Shaoyun; Zhang, Xu; Li, Changyi

    2016-01-01

    Objectives The formation of biofilm around implants, which is induced by immediate bacterial colonization after installation, is the primary cause of post-operation infection. Initial surface modification is usually required to incorporate antibacterial agents on titanium (Ti) surfaces to inhibit biofilm formation. However, simple and effective priming methods are still lacking for the development of an initial functional layer as a base for subsequent coatings on titanium surfaces. The purpose of our work was to establish a novel initial layer on Ti surfaces using phase-transited lysozyme (PTL), on which multilayer coatings can incorporate silver nanoparticles (AgNP) using chitosan (CS) and hyaluronic acid (HA) via a layer-by-layer (LbL) self-assembly technique. Methods In this study, the surfaces of Ti substrates were primed by dipping into a mixture of lysozyme and tris(2-carboxyethyl)phosphine (TCEP) to obtain PTL-functionalized Ti substrates. The subsequent alternating coatings of HA and chitosan loaded with AgNP onto the precursor layer of PTL were carried out via LbL self-assembly to construct multilayer coatings on Ti substrates. Results The results of SEM and XPS indicated that the necklace-like PTL and self-assembled multilayer were successfully immobilized on the Ti substrates. The multilayer coatings loaded with AgNP can kill planktonic and adherent bacteria to 100% during the first 4 days. The antibacterial efficacy of the samples against planktonic and adherent bacteria achieved 65%-90% after 14 days. The sustained release of Ag over 14 days can prevent bacterial invasion until mucosa healing. Although the AgNP-containing structure showed some cytotoxicity, the toxicity can be reduced by controlling the Ag release rate and concentration. Conclusions The PTL priming method provides a promising strategy for fabricating long-term antibacterial multilayer coatings on titanium surfaces via the LbL self-assembly technique, which is effective in preventing

  4. Protein imprinted ionic liquid polymer on the surface of multiwall carbon nanotubes with high binding capacity for lysozyme.

    PubMed

    Yuan, Shifang; Deng, Qiliang; Fang, Guozhen; Wu, Jianhua; Li, Wangwang; Wang, Shuo

    2014-06-01

    In this research, ionic liquid as functional monomer to prepare molecularly imprinted polymers for protein recognition was for the first time demonstrated, in which, 1-vinyl-3-butylimidazolium chloride was selected as functional monomer, acrylamide as co-functional monomer and lysozyme (Lyz) as template protein to synthesize imprinted polymers on the surface of multiwall carbon nanotubes in aqueous medium. The results indicated that ionic liquid was helpful to improve binding capacity of imprinted polymers, which had a maximum binding capacity of 763.36 mg/g in the optimum adsorption conditions. The prepared imprinted polymers had a fast adsorption rate and a much higher adsorption capacity than the corresponding non-imprinted polymers, with the difference in adsorption capacity up to 258.31 mg/g. The obtained polymer was evaluated by Lyz, bovine serum albumin (BSA), bovine hemoglobin (BHb), equine myoglobin (MB) and cytochrome c (Cyt c). The selectivity factor (β) for Lyz/BSA, Lyz/Mb, Lyz/BHb, and Lyz/Cyt c were 7.17, 2.12, 1.76 and 1.57, respectively, indicating the imprinted polymers had a good selectivity. Easy preparation of the imprinted polymers as well as high ability and selectivity to adsorb template proteins makes this polymer attractive and broadly applicable in biomacromolecular separation, biotechnology and sensors.

  5. Mixed-mode chromatography integrated with high-performance liquid chromatography for protein analysis and separation: Using bovine serum albumin and lysozyme as the model target.

    PubMed

    Xia, Hai-Feng; Don, Bin-Bin; Zheng, Meng-Jie

    2016-05-01

    A type of mixed-mode chromatography was integrated with high-performance liquid chromatography for protein analysis and separation. The chromatographic behavior was tested using bovine serum albumin and lysozyme as model proteins. For the mixed-mode column, the silica beads were activated with γ-(2,3-epoxypropoxy)-propytrimethoxysilane and coupled with 4-mercaptopyridine as the functional ligand. The effects of pH, salt, and the organic solvent conditions of the mobile phase on the retention behavior were studied, which provided valuable clues for separation strategy. When eluted with a suitable pH gradient, salt concentration gradient, and acetonitrile content gradient, the separation behavior of bovine serum albumin and lysozyme could be controlled by altering the conditions of the mobile phase. The results indicated this type of chromatography might be a useful method for protein analysis and separation.

  6. Lysozyme separation by hollow-fibre ultrafiltration.

    PubMed

    Ghosh; Silva1; Cui

    2000-08-01

    This paper discusses the purification of lysozyme from chicken egg white using hollow-fibre ultrafiltration (30kDa MWCO, polysulphone membrane). Lysozyme is preferentially transmitted through the membrane while the membrane largely retains other egg white proteins. Improvement in system hydrodynamics resulted in an increase in permeate flux while lysozyme transmission remained unaffected, leading to higher productivity. The percentage purity of lysozyme obtained was generally insensitive to system hydrodynamics. The permeate flux and productivity increased with increase in transmembrane pressure (TMP) before levelling off around 0.7bar. However, the TMP did not have any pronounced effect on the transmission and the purity of lysozyme. Experiments carried out in the diafiltration mode showed that moderately pure lysozyme (80-90%) could be obtained in an extended operation.

  7. Inactivation of indispensable bacterial proteins by early proteins of bacteriophages: implication in antibacterial drug discovery.

    PubMed

    Sau, S; Chattoraj, P; Ganguly, T; Chanda, P K; Mandal, N C

    2008-06-01

    Bacteriophages utilize host bacterial cellular machineries for their own reproduction and completion of life cycles. The early proteins that phage synthesize immediately after the entry of their genomes into bacterial cells participate in inhibiting host macromolecular biosynthesis, initiating phage-specific replication and synthesizing late proteins. Inhibition of synthesis of host macromolecules that eventually leads to cell death is generally performed by the physical and/or chemical modification of indispensable host proteins by early proteins. Interestingly, most modified bacterial proteins were shown to take part actively in phage-specific transcription and replication. Research on phages in last nine decades has demonstrated such lethal early proteins that interact with or chemically modify indispensable host proteins. Among the host proteins inhibited by lethal phage proteins, several are not inhibited by any chemical inhibitor available today. Under the context of widespread dissemination of antibiotic-resistant strains of pathogenic bacteria in recent years, the information of lethal phage proteins and cognate host proteins could be extremely invaluable as they may lead to the identification of novel antibacterial compounds. In this review, we summarize the current knowledge about some early phage proteins, their cognate host proteins and their mechanism of action and also describe how the above interacting proteins had been exploited in antibacterial drug discovery.

  8. Influence of core and maltose surface modification of PEIs on their interaction with plasma proteins-Human serum albumin and lysozyme.

    PubMed

    Wrobel, Dominika; Marcinkowska, Monika; Janaszewska, Anna; Appelhans, Dietmar; Voit, Brigitte; Klajnert-Maculewicz, Barbara; Bryszewska, Maria; Štofik, Marcel; Herma, Regina; Duchnowicz, Piotr; Maly, Jan

    2017-04-01

    Regardless of the route of administration, some or all of a therapeutic agent will appear in the blood stream, where it can act on blood cells and other components of the plasma. Recently we have shown that poly(ethylene imines) (PEIs) which interact with plasma proteins are taken up into erythrocyte membranes. These observations led us to investigate the interactions between maltose functionalized hyperbranched PEIs (PEI-Mal) and plasma proteins. Two model proteins were chosen - human serum albumin (HSA) (albumins constitute ∼60% of all plasma proteins), and lysozyme. HSA is a negatively charged 66kDa protein at neutral pH, whereas lysozyme is a positively charged 14kDa protein. Fluorescence quenching and changes in the conformation of the amino acid tryptophan, diameter and zeta potential of proteins were investigated to evaluate the interaction of PEI-Mal with proteins. PEI-Mal interacts with both types of proteins. The strength of dendritic glycopolymer interactions was generally weak, especially with lysozyme. Greater changes were found with HSA, mainly triggered by hydrogen bonds and the electrostatic interaction properties of dendritic glycopolymers. Moreover, the structure and the size of PEI-Mal macromolecules affected these interactions; larger macromolecules with more sugar groups (95% maltose units) interacted more strongly with proteins than smaller ones with lower sugar modification (33% maltose units). Due to (i) the proven overall low toxicity of sugar-modified PEIs and, (ii) their ability to interact preferentially through hydrogen bonds with proteins of human plasma or possibly with other interesting protein targets, PEI-Mal is a good candidate for creating therapeutic nanoparticles in the fast developing field of nanomedicine.

  9. Mass spectrometry-based proteomics of oxidative stress: Identification of 4-hydroxy-2-nonenal (HNE) adducts of amino acids using lysozyme and bovine serum albumin as model proteins.

    PubMed

    Aslebagh, Roshanak; Pfeffer, Bruce A; Fliesler, Steven J; Darie, Costel C

    2016-10-01

    Modification of proteins by 4-hydroxy-2-nonenal (HNE), a reactive by-product of ω6 polyunsaturated fatty acid oxidation, on specific amino acid residues is considered a biomarker for oxidative stress, as occurs in many metabolic, hereditary, and age-related diseases. HNE modification of amino acids can occur either via Michael addition or by formation of Schiff-base adducts. These modifications typically occur on cysteine (Cys), histidine (His), and/or lysine (Lys) residues, resulting in an increase of 156 Da (Michael addition) or 138 Da (Schiff-base adducts), respectively, in the mass of the residue. Here, we employed biochemical and mass spectrometry (MS) approaches to determine the MS "signatures" of HNE-modified amino acids, using lysozyme and BSA as model proteins. Using direct infusion of unmodified and HNE-modified lysozyme into an electrospray quadrupole time-of-flight mass spectrometer, we were able to detect up to seven HNE modifications per molecule of lysozyme. Using nanoLC-MS/MS, we found that, in addition to N-terminal amino acids, Cys, His, and Lys residues, HNE modification of arginine (Arg), threonine (Thr), tryptophan (Trp), and histidine (His) residues can also occur. These sensitive and specific methods can be applied to the study of oxidative stress to evaluate HNE modification of proteins in complex mixtures from cells and tissues under diseased versus normal conditions.

  10. Antioxidant, Antibacterial, and Cytoprotective Activity of Agathi Leaf Protein

    PubMed Central

    Zarena, A. S.; Gopal, Shubha; Vineeth, R.

    2014-01-01

    In the present study a protein termed agathi leaf protein (ALP) from Sesbania grandiflora Linn. (agathi) leaves was isolated after successive precipitation with 65% ammonium sulphate followed by purification on Sephadex G 75. The column chromatography of the crude protein resulted in four peaks of which Peak I (P I) showed maximum inhibition activity against hydroxyl radical. SDS-PAGE analysis of P I indicated that the molecular weight of the protein is ≈29 kDa. The purity of the protein was 98.4% as determined by RP-HPLC and showed a single peak with a retention time of 19.9 min. ALP was able to reduce oxidative damage by scavenging lipid peroxidation against erythrocyte ghost (85.50 ± 6.25%), linolenic acid (87.67 ± 3.14%) at 4.33 μM, ABTS anion (88 ± 3.22%), and DNA damage (83 ± 4.20%) at 3.44 μM in a dose-dependent manner. The purified protein offered significant protection to lymphocyte (72% at 30 min) induced damage by t-BOOH. In addition, ALP showed strong antibacterial activity against Pseudomonas aeruginosa (20 ± 3.64 mm) and Staphylococcus aureus (19 ± 1.53 mm) at 200 μg/mL. The safety assessment showed that ALP does not induce cytotoxicity towards human lymphocyte at the tested concentration of 0.8 mg/mL. PMID:24616824

  11. The synthesis of magnetic lysozyme-imprinted polymers by means of distillation-precipitation polymerization for selective protein enrichment.

    PubMed

    Cao, Jiali; Zhang, Xihao; He, Xiwen; Chen, Langxing; Zhang, Yukui

    2014-02-01

    A protein imprinting approach for the synthesis of core-shell structure nanoparticles with a magnetic core and molecularly imprinted polymer (MIP) shell was developed using a simple distillation-precipitation polymerization method. In this work, Fe3O4 magnetic nanoparticles were first synthesized through a solvothermal method and then were conveniently surface-modified with 3-(methacryloyloxy)propyltrimethoxylsilane as anchor molecules to donate vinyl groups. Next a high-density MIP shell was coated onto the surface of the magnetic nanoparticles by the copolymerization of functional monomer acrylamide (AAm), cross-linking agent N,N'-methylenebisacrylamide (MBA), the initiator azodiisobutyronitrile (AIBN), and protein in acetonitrile heated at reflux. The morphology, adsorption, and recognition properties of the magnetic molecularly imprinted nanoparticles were investigated by transmission electron microscopy (TEM), scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), vibrating sample magnetometer (VSM), and rebinding experiments. The resulting MIP showed a high adsorption capacity (104.8 mg g(-1)) and specific recognition (imprinting factor=7.6) to lysozyme (Lyz). The as-prepared Fe3O4@Lyz-MIP nanoparticles with a mean diameter of 320 nm were coated with an MIP shell that was 20 nm thick, which enabled Fe3O4@Lyz-MIP to easily reach adsorption equilibrium. The high magnetization saturation (40.35 emu g(-1)) endows the materials with the convenience of magnetic separation under an external magnetic field and allows them to be subsequently reused. Furthermore, Fe3O4@Lyz-MIP could selectively extract a target protein from real egg-white samples under an external magnetic field.

  12. A novel antifungal protein with lysozyme-like activity from seeds of Clitoria ternatea.

    PubMed

    K, Ajesh; K, Sreejith

    2014-06-01

    An antifungal protein with a molecular mass of 14.3 kDa was isolated from the seeds of butterfly pea (Clitoria ternatea) and designated as Ct protein. The antifungal protein was purified using different methods including ammonium sulphate precipitation, ion exchange chromatography on DEAE-cellulose and gel filtration on Sephadex G-50 column. Ct protein formed a single colourless rod-shaped crystal by hanging drop method after 7 days of sample loading. The protein showed lytic activity against Micrococcus luteus and broad-spectrum, fungicidal activity, particularly against the most clinically relevant yeasts, such as Cryptococcus neoformans, Cryptococcus albidus, Cryptococcus laurentii, Candida albicans and Candida parapsilosis. It also exerted an inhibitory activity on mycelial growth in several mould species including Curvularia sp., Alternaria sp., Cladosporium sp., Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Rhizopus sp., and Sclerotium sp. The present study adds to the literature on novel seed proteins with antifungal activity.

  13. Proteins in vacuo: A molecular dynamics study of the unfolding behavior of highly charged disulfide-bond-intact lysozyme subjected to a temperature pulse

    NASA Astrophysics Data System (ADS)

    Reimann, C. T.; Velázquez, I.; Bittner, M.; Tapia, O.

    1999-12-01

    Molecular dynamics simulations were used to interpret a variety of experimental data on highly charged disulfide-bond-intact lysozyme in vacuo. The simulation approach involved submitting a model of the protein [Reimann, Velázquez, and Tapia, J. Phys. Chem. B 102, 9344 (1998)] in a given charge state to a 3-ns-long heat pulse (usually at 500 K) followed by cooling or relaxation for 1 ns back to room temperature (293 K). This treatment yielded a charge threshold around Q0=8+ for obtaining significant unfolding, as indicated by an enhancement in collision cross section and conformer length. The collision cross sections and lengths theoretically obtained, along with the threshold charge state for initiating unfolding, were compatible with experimental results on lysozyme in vacuo. The unfolded, highly elongated conformations obtained for Q>=9+ displayed a significant level of non-native β-sheet content which appeared to be additionally stabilized by charge self-solvation.

  14. A bifunctional invertebrate-type lysozyme from the disk abalone, Haliotis discus discus: genome organization, transcriptional profiling and biological activities of recombinant protein.

    PubMed

    Bathige, S D N K; Umasuthan, Navaneethaiyer; Kasthuri, Saranya Revathy; Whang, Ilson; Lim, Bong-Soo; Nam, Bo-Hye; Lee, Jehee

    2013-10-01

    Lysozyme is an important enzyme in the innate immune system that plays a vital role in fighting microbial infections. In the current study, we identified, cloned, and characterized a gene that encodes an invertebrate-type lysozyme from the disk abalone, Haliotis discus discus (abLysI). The full-length cDNA of abLysI consisted of 545 bp with an open reading frame of 393 bp that encodes 131 amino acids. The theoretical molecular mass of mature abLysI was 12.3 kDa with an isoelectric point of 8.03. Conserved features in other homologs, such as catalytic sites for lytic activity (Glu(30) and Asp(41)), isopeptidase activity (His(107)), and ten cysteine residues were identified in abLysI. Genomic sequence analysis with respect to its cDNA showed that abLysI was organized into four exons interrupted by three introns. Several immune-related transcription factor binding sites were discovered in the putative promoter region. Homology and phylogeny analysis of abLysI depicted high identity and closer proximity, respectively, with an annelid i-type lysozyme from Hirudo medicinalis, and indicated that abLysI is a novel molluscan i-type lysozyme. Tissue-specific expressional studies revealed that abLysI is mainly transcribed in hepatopancreas followed by mantle. In addition, abLysI mRNA expression was induced following bacterial (Vibrio parahaemolyticus and Listeria monocytogenes) and viral (viral hemorrhagic septicemia virus) challenges. Recombinantly expressed abLysI [(r)abLysI] demonstrated strong lytic activity against Micrococcus lysodeikticus, isopeptidase activity, and antibacterial activity against several Gram-positive and Gram-negative bacteria. Moreover, (r)abLysI showed optimum lytic activity at pH 4.0 and 60 °C, while exhibiting optimum isopeptidase activity at pH 7.0. Taken together, these results indicate that abLysI is potentially involved in immune responses of the disk abalone to protect it from invaders.

  15. A study of renaturation of reduced hen egg white lysozyme. Enzymically active intermediates formed during oxidation of the reduced protein.

    PubMed

    Acharya, A S; Taniuchi, H

    1976-11-25

    The material obtained from reduced hen egg white lysozyme after complete air oxidation at pH 8.0 and 37 degrees has yielded, by gel filtration on a Bio-Gel P-30 column, enzymically active species and an enzymically inactive form which eluted sooner than the active species but later than expected for a dimer of lysozyme. Reduced lysozyme also elutes at the same position as this inactive material. Examination of the fragments produced on CNBr cleavage of the inactive form indicates that at least 24% of the population contains incorrect disulfide bonds involving half-cystine residues 6, 30, 115, and 127. Tryptophan fluorescence and the intrinsic viscosity of the inactive form show an enlarged molecular domain with a disordered conformation. The yield of the inactive form increases as the oxidation of reduced lysozyme is accelerated using cupric ion. In the presence of 4 X 10(-5) M cupric ion, reduced lysozyme forms almost quantitatively the inactive form, which is almost completely converted to the native form by sulfhydryl-disulfide interchange catalyzed by thiol groups of either reduced lysozyme or beta-mercaptoethanol. The material trapped by alkylation of the free sulfhydryl groups with [1-14C]iodoacetic acid during the early stage of air oxidation of reduced lysozyme was fractionated by gel filtration to permit separation of the active species from the inactive form. Ion exchange chromatography of the active species yielded completely renatured lysozyme and three major enzymically active radioactive derivatives. Two of these derivatives contained approximately 2 mol of S-carboxymethylcysteine. Isolation and characterization of radioactive tryptic peptides from each of the three active forms, permitted the identification of Cys 6 and Cys 127, Cys 76 and 94, and Cys 80 as the sulfhydryl groups alkylated in these three incompletely oxidized, partially active forms. Thus, it appears that the interatomic interactions maintaining the compact three-dimensional structure

  16. Covalent attachment of lysozyme to cotton/cellulose materials: protein verses solid support activation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Covalent attachment of enzymes to cellulosic materials like cotton is of interest where either release or loss of enzyme activity over time needs to be avoided. The covalent attachment of an enzyme to a cellulosic substrate requires either activation of a protein side chain or an organic functional ...

  17. Molecular Dynamics Analysis of Lysozyme Protein in Ethanol- Water Mixed Solvent

    DTIC Science & Technology

    2012-01-01

    The general formula for ethanol is given as C2H5OH ( Burkhard & Dennison, 1951). It is colorless, volatile and flammable liquid. The physical...structure prediction. PloS one, 4(6), e5840. Branden, C., & Tooze, J. (1991). Introduction to protein structure (Vol. 2): Garland New York. Burkhard , D

  18. Purification and Characterization of Recombinant Human Lysozyme from Eggs of Transgenic Chickens.

    PubMed

    Wu, Hanyu; Cao, Dainan; Liu, Tongxin; Zhao, Jianmin; Hu, Xiaoxiang; Li, Ning

    2015-01-01

    Transgenic chickens as bioreactors have several advantages, such as the simple establishment procedure, correct glycosylation profile of expressed proteins, etc. Lysozyme is widely used in food industry, livestock farming, and medical field as a replacement of antibiotics because of its antibacterial and complement system-modulating activity. In this study, we used RT-PCR, Western blot, and immunofluorescence to detect the expression of recombinant human lysozyme (rhLY) in the transgenic chicken. We demonstrated that the transgene of rhLY was genetically stable across different generations. We next optimized the purification procedure of rhLY from the transgenic eggs by utilizing two steps of cation-exchange chromatography and one gel-filtration chromatography. About 6 mg rhLY with the purity exceeding 90% was obtained from ten eggs, and the purification efficiency was about 75%. The purified rhLY had similar physicochemical and biological properties in molecular mass and antibacterial activity compared to the commercial human lysozyme. Additionally, both of them exhibited thermal stability at 60°C and tolerated an extensive pH range of 2 to 11. In conclusion, our study proved that the transgenic chickens we have previously generated were genetically stable and suitable for the production of active rhLY. We also provided a pipeline for purifying the recombinant proteins from transgenic eggs, which could be useful for other studies.

  19. Comparison of bactericidal activity of six lysozymes at atmospheric pressure and under high hydrostatic pressure.

    PubMed

    Nakimbugwe, Dorothy; Masschalck, Barbara; Atanassova, Miroslava; Zewdie-Bosüner, Abebetch; Michiels, Chris W

    2006-05-01

    The antibacterial working range of six lysozymes was tested under ambient and high pressure, on a panel of five gram-positive (Enterococcus faecalis, Bacillus subtilis, Listeria innocua, Staphylococcus aureus and Micrococcus lysodeikticus) and five gram-negative bacteria (Yersinia enterocolitica, Shigella flexneri, Escherichia coli O157:H7, Pseudomonas aeruginosa and Salmonella typhimurium). The lysozymes included two that are commercially available (hen egg white lysozyme or HEWL, and mutanolysin from Streptomyces globisporus or M1L), and four that were chromatographically purified (bacteriophage lambda lysozyme or LaL, bacteriophage T4 lysozyme or T4L, goose egg white lysozyme or GEWL, and cauliflower lysozyme or CFL). T4L, LaL and GEWL were highly pure as evaluated by silver staining of SDS-PAGE gels and zymogram analysis while CFL was only partially pure. At ambient pressure each gram-positive test organism displayed a specific pattern of sensitivity to the six lysozymes, but none of the gram-negative bacteria was sensitive to any of the lysozymes. High pressure treatment (130-300 MPa, 25 degrees C, 15 min) sensitised several gram-positive and gram-negative bacteria for one or more lysozymes. M. lysodeikticus and P. aeruginosa became sensitive to all lysozymes under high pressure, S. typhimurium remained completely insensitive to all lysozymes, and the other bacteria showed sensitisation to some of the lysozymes. The possible applications of the different lysozymes as biopreservatives, and the possible reasons for the observed differences in bactericidal specificity are discussed.

  20. Galleria mellonella lysozyme induces apoptotic changes in Candida albicans cells.

    PubMed

    Sowa-Jasiłek, Aneta; Zdybicka-Barabas, Agnieszka; Stączek, Sylwia; Wydrych, Jerzy; Skrzypiec, Krzysztof; Mak, Paweł; Deryło, Kamil; Tchórzewski, Marek; Cytryńska, Małgorzata

    2016-12-01

    The greater wax moth Galleria mellonella has been increasingly used as a model host to determine Candida albicans virulence and efficacy of antifungal treatment. The G. mellonella lysozyme, similarly to its human counterpart, is a member of the c-type family of lysozymes that exhibits antibacterial and antifungal activity. However, in contrast to the relatively well explained bactericidal action, the mechanism of fungistatic and/or fungicidal activity of lysozymes is still not clear. In the present study we provide the direct evidences that the G. mellonella lysozyme binds to the protoplasts as well as to the intact C. albicans cells and decreases the survival rate of both these forms in a time-dependent manner. No enzymatic activity of the lysozyme towards typical chitinase and β-glucanase substrates was detected, indicating that hydrolysis of main fungal cell wall components is not responsible for anti-Candida activity of the lysozyme. On the other hand, pre-treatment of cells with tetraethylammonium, a potassium channel blocker, prevented them from the lysozyme action, suggesting that lysozyme acts by induction of programmed cell death. In fact, the C. albicans cells treated with the lysozyme exhibited typical apoptotic features, i.e. loss of mitochondrial membrane potential, phosphatidylserine exposure in the outer leaflet of the cell membrane, as well as chromatin condensation and DNA fragmentation.

  1. The level of some acute phase proteins, total protein, gamma-globulins and activity of lysozyme in blood plasma of rats supplemented with vitamin E and exposed to ozone.

    PubMed

    Jakubowski, K; Jedlińska-Krakowska, M; Siwicki, A K

    2004-01-01

    In rats exposed for 28 days (5 hours a day) to ozone at a concentration of 0.5 ppm and receiving alpha-tocopherol at doses of 4.5 mg/rat and 15 mg/rat, levels of acute phase proteins (APP)--C-reactive protein (CRP), ceruloplasmin (Cp), total protein, gamma-globulins, and activity of lysozyme in blood serum were studied. The assays were performed in the presence of respective control groups, i.e. rats receiving the same doses of alpha-tocopherol but not exposed to ozone, a group of animals not supplemented with vitamin but exposed to ozone, a group of animals injected with physiological fluid and a control group not subjected to any of the treatments. The study revealed that the ozone-exposed animals had an increased lysozyme activity and a decreased total protein level. However, in rats protected by alpha-tocopherol and exposed to ozone, the concentration of APP, lysozyme activity and total protein were found to be decreased. Similar relationships also occurred in animals receiving alpha-tocopherol and not exposed to ozone.

  2. Protein disulfide isomerase-P5, down-regulated in the final stage of boar epididymal sperm maturation, catalyzes disulfide formation to inhibit protein function in oxidative refolding of reduced denatured lysozyme.

    PubMed

    Akama, Kuniko; Horikoshi, Tomoe; Sugiyama, Atsushi; Nakahata, Satoko; Akitsu, Aoi; Niwa, Nobuyoshi; Intoh, Atsushi; Kakui, Yasutaka; Sugaya, Michiko; Takei, Kazuo; Imaizumi, Noriaki; Sato, Takaya; Matsumoto, Rena; Iwahashi, Hitoshi; Kashiwabara, Shin-ichi; Baba, Tadashi; Nakamura, Megumi; Toda, Tosifusa

    2010-06-01

    In mammalian spermiogenesis, sperm mature during epididymal transit to get fertility. The pig sharing many physiological similarities with humans is considered a promising animal model in medicine. We examined the expression profiles of proteins from boar epididymal caput, corpus, and cauda sperm by two-dimensional gel electrophoresis and peptide mass fingerprinting. Our results indicated that protein disulfide isomerase-P5 (PDI-P5) human homolog was down-regulated from the epididymal corpus to cauda sperm, in contrast to the constant expression of protein disulfide isomerase A3 (PDIA3) human homolog. To examine the functions of PDIA3 and PDI-P5, we cloned and sequenced cDNAs of pig PDIA3 and PDI-P5 protein precursors. Each recombinant pig mature PDIA3 and PDI-P5 expressed in Escherichia coli showed thiol-dependent disulfide reductase activities in insulin turbidity assay. Although PDIA3 showed chaperone activity to promote oxidative refolding of reduced denatured lysozyme, PDI-P5 exhibited anti-chaperone activity to inhibit oxidative refolding of lysozyme at an equimolar ratio. SDS-PAGE and Western blotting analysis suggested that disulfide cross-linked and non-productively folded lysozyme was responsible for the anti-chaperone activity of PDI-P5. These results provide a molecular basis and insights into the physiological roles of PDIA3 and PDI-P5 in sperm maturation and fertilization.

  3. Co-option of bacteriophage lysozyme genes by bivalve genomes

    PubMed Central

    Wang, Chunyang; Jin, Min; Lan, Jiangfeng; Ye, Ting; Hui, Kaimin; Tan, Jingmin; Wang, Zheng; Wang, Wen; Han, Guan-Zhu

    2017-01-01

    Eukaryotes have occasionally acquired genetic material through horizontal gene transfer (HGT). However, little is known about the evolutionary and functional significance of such acquisitions. Lysozymes are ubiquitous enzymes that degrade bacterial cell walls. Here, we provide evidence that two subclasses of bivalves (Heterodonta and Palaeoheterodonta) acquired a lysozyme gene via HGT, building on earlier findings. Phylogenetic analyses place the bivalve lysozyme genes within the clade of bacteriophage lysozyme genes, indicating that the bivalves acquired the phage-type lysozyme genes from bacteriophages, either directly or through intermediate hosts. These bivalve lysozyme genes underwent dramatic structural changes after their co-option, including intron gain and fusion with other genes. Moreover, evidence suggests that recurrent gene duplication occurred in the bivalve lysozyme genes. Finally, we show the co-opted lysozymes exhibit a capacity for antibacterial action, potentially augmenting the immune function of related bivalves. This represents an intriguing evolutionary strategy in the eukaryote–microbe arms race, in which the genetic materials of bacteriophages are co-opted by eukaryotes, and then used by eukaryotes to combat bacteria, using a shared weapon against a common enemy. PMID:28100665

  4. A class of selective antibacterials derived from a protein kinase inhibitor pharmacophore.

    PubMed

    Miller, J Richard; Dunham, Steve; Mochalkin, Igor; Banotai, Craig; Bowman, Matthew; Buist, Susan; Dunkle, Bill; Hanna, Debra; Harwood, H James; Huband, Michael D; Karnovsky, Alla; Kuhn, Michael; Limberakis, Chris; Liu, Jia Y; Mehrens, Shawn; Mueller, W Thomas; Narasimhan, Lakshmi; Ogden, Adam; Ohren, Jeff; Prasad, J V N Vara; Shelly, John A; Skerlos, Laura; Sulavik, Mark; Thomas, V Hayden; VanderRoest, Steve; Wang, LiAnn; Wang, Zhigang; Whitton, Amy; Zhu, Tong; Stover, C Kendall

    2009-02-10

    As the need for novel antibiotic classes to combat bacterial drug resistance increases, the paucity of leads resulting from target-based antibacterial screening of pharmaceutical compound libraries is of major concern. One explanation for this lack of success is that antibacterial screening efforts have not leveraged the eukaryotic bias resulting from more extensive chemistry efforts targeting eukaryotic gene families such as G protein-coupled receptors and protein kinases. Consistent with a focus on antibacterial target space resembling these eukaryotic targets, we used whole-cell screening to identify a series of antibacterial pyridopyrimidines derived from a protein kinase inhibitor pharmacophore. In bacteria, the pyridopyrimidines target the ATP-binding site of biotin carboxylase (BC), which catalyzes the first enzymatic step of fatty acid biosynthesis. These inhibitors are effective in vitro and in vivo against fastidious gram-negative pathogens including Haemophilus influenzae. Although the BC active site has architectural similarity to those of eukaryotic protein kinases, inhibitor binding to the BC ATP-binding site is distinct from the protein kinase-binding mode, such that the inhibitors are selective for bacterial BC. In summary, we have discovered a promising class of potent antibacterials with a previously undescribed mechanism of action. In consideration of the eukaryotic bias of pharmaceutical libraries, our findings also suggest that pursuit of a novel inhibitor leads for antibacterial targets with active-site structural similarity to known human targets will likely be more fruitful than the traditional focus on unique bacterial target space, particularly when structure-based and computational methodologies are applied to ensure bacterial selectivity.

  5. A class of selective antibacterials derived from a protein kinase inhibitor pharmacophore

    SciTech Connect

    Miller, J. Richard; Dunham, Steve; Mochalkin, Igor; Banotai, Craig; Bowman, Matthew; Buist, Susan; Dunkle, Bill; Hanna, Debra; Harwood, H. James; Huband, Michael D.; Karnovsky, Alla; Kuhn, Michael; Limberakis, Chris; Liu, Jia Y.; Mehrens, Shawn; Mueller, W. Thomas; Narasimhan, Lakshmi; Ogden, Adam; Ohren, Jeff; Prasad, J.V.N. Vara; Shelly, John A.; Skerlos, Laura; Sulavik, Mark; Thomas, V. Hayden; VanderRoest, Steve; Wang, LiAnn; Wang, Zhigang; Whitton, Amy; Zhu, Tong; Stover, C. Kendall

    2009-06-25

    As the need for novel antibiotic classes to combat bacterial drug resistance increases, the paucity of leads resulting from target-based antibacterial screening of pharmaceutical compound libraries is of major concern. One explanation for this lack of success is that antibacterial screening efforts have not leveraged the eukaryotic bias resulting from more extensive chemistry efforts targeting eukaryotic gene families such as G protein-coupled receptors and protein kinases. Consistent with a focus on antibacterial target space resembling these eukaryotic targets, we used whole-cell screening to identify a series of antibacterial pyridopyrimidines derived from a protein kinase inhibitor pharmacophore. In bacteria, the pyridopyrimidines target the ATP-binding site of biotin carboxylase (BC), which catalyzes the first enzymatic step of fatty acid biosynthesis. These inhibitors are effective in vitro and in vivo against fastidious Gram-negative pathogens including Haemophilus influenzae. Although the BC active site has architectural similarity to those of eukaryotic protein kinases, inhibitor binding to the BC ATP-binding site is distinct from the protein kinase-binding mode, such that the inhibitors are selective for bacterial BC. In summary, we have discovered a promising class of potent antibacterials with a previously undescribed mechanism of action. In consideration of the eukaryotic bias of pharmaceutical libraries, our findings also suggest that pursuit of a novel inhibitor leads for antibacterial targets with active-site structural similarity to known human targets will likely be more fruitful than the traditional focus on unique bacterial target space, particularly when structure-based and computational methodologies are applied to ensure bacterial selectivity.

  6. Protonation of interacting residues in a protein by a Monte Carlo method: application to lysozyme and the photosynthetic reaction center of Rhodobacter sphaeroides.

    PubMed Central

    Beroza, P; Fredkin, D R; Okamura, M Y; Feher, G

    1991-01-01

    We used Monte Carlo methods to treat statistical problem of electrostatic interactions among many titrating amino acids and applied these methods to lysozyme and the photosynthetic reaction center of Rhodobacter sphaeroides, including all titrating sites. We computed the average protonation of residues as a function of pH from an equilibrium distribution of states generated by random sampling. Electrostatic energies were calculated from a finite difference solution to the linearized Poisson-Boltzmann equation using the coordinates from solved protein structures. For most calculations we used the Metropolis algorithm to sample protonation states; for strongly coupled sites, we substantially reduced sampling errors by using a modified algorithm that allows multiple site transitions. The Monte Carlo method agreed with calculations for a small test system, lysozyme, for which the complete partition function was calculated. We also calculated the pH dependence of the free energy change associated with electron transfer from the primary to the secondary quinone in the photosynthetic reaction center. The shape of the resulting curve agreed fairly well with experiment, but the proton uptake from which the free energy was calculated agreed only to within a factor of two with the observed values. We believe that this discrepancy resulted from errors in the individual electrostatic energy calculations rather than from errors in the Monte Carlo sampling. PMID:2062860

  7. A test of the "jigsaw puzzle" model for protein folding by multiple methionine substitutions within the core of T4 lysozyme.

    PubMed Central

    Gassner, N C; Baase, W A; Matthews, B W

    1996-01-01

    To test whether the structure of a protein is determined in a manner akin to the assembly of a jigsaw puzzle, up to 10 adjacent residues within the core of T4 lysozyme were replaced by methionine. Such variants are active and fold cooperatively with progressively reduced stability. The structure of a seven-methionine variant has been shown, crystallographically, to be similar to wild type and to maintain a well ordered core. The interaction between the core residues is, therefore, not strictly comparable with the precise spatial complementarity of the pieces of a jigsaw puzzle. Rather, a certain amount of give and take in forming the core structure is permitted. A simplified hydrophobic core sequence, imposed without genetic selection or computer-based design, is sufficient to retain native properties in a globular protein. Images Fig. 2 Fig. 3 PMID:8901549

  8. Negative protein 1, which is required for function of the chicken lysozyme gene silencer in conjunction with hormone receptors, is identical to the multivalent zinc finger repressor CTCF.

    PubMed Central

    Burcin, M; Arnold, R; Lutz, M; Kaiser, B; Runge, D; Lottspeich, F; Filippova, G N; Lobanenkov, V V; Renkawitz, R

    1997-01-01

    The transcriptional repressor negative protein 1 (NeP1) binds specifically to the F1 element of the chicken lysozyme gene silencer and mediates synergistic repression by v-ERBA, thyroid hormone receptor, or retinoic acid receptor. Another protein, CCCTC-binding factor (CTCF), specifically binds to 50-bp-long sequences that contain repetitive CCCTC elements in the vicinity of vertebrate c-myc genes. Previously cloned chicken, mouse, and human CTCF cDNAs encode a highly conserved 11-Zn-finger protein. Here, NeP1 was purified and DNA bases critical for NeP1-F1 interaction were determined. NeP1 is found to bind a 50-bp stretch of nucleotides without any obvious sequence similarity to known CTCF binding sequences. Despite this remarkable difference, these two proteins are identical. They have the same molecular weight, and NeP1 contains peptide sequences which are identical to sequences in CTCF. Moreover, NeP1 and CTCF specifically recognize each other's binding DNA sequence and induce identical conformational alterations in the F1 DNA. Therefore, we propose to replace the name NeP1 with CTCF. To analyze the puzzling sequence divergence in CTCF binding sites, we studied the DNA binding of 12 CTCF deletions with serially truncated Zn fingers. While fingers 4 to 11 are indispensable for CTCF binding to the human c-myc P2 promoter site A, a completely different combination of fingers, namely, 1 to 8 or 5 to 11, was sufficient to bind the lysozyme silencer site F1. Thus, CTCF is a true multivalent factor with multiple repressive functions and multiple sequence specificities. PMID:9032255

  9. Protein crowding in solution, frozen and freeze-dried states: small-angle neutron and X-ray scattering study of lysozyme/sorbitol/water systems

    NASA Astrophysics Data System (ADS)

    Krueger, Susan; Khodadadi, Sheila; Clark, Nicholas; McAuley, Arnold; Cristiglio, Viviana; Theyencheri, Narayanan; Curtis, Joseph; Shalaev, Evgenyi

    2015-03-01

    For effective preservation, proteins are often stored as frozen solutions or in glassy states using a freeze-drying process. However, aggregation is often observed after freeze-thaw or reconstitution of freeze-dried powder and the stability of the protein is no longer assured. In this study, small-angle neutron and X-ray scattering (SANS and SAXS) have been used to investigate changes in protein-protein interaction distances of a model protein/cryoprotectant system of lysozyme/sorbitol/water, under representative pharmaceutical processing conditions. The results demonstrate the utility of SAXS and SANS methods to monitor protein crowding at different stages of freezing and drying. The SANS measurements of solution samples showed at least one protein interaction peak corresponding to an interaction distance of ~ 90 Å. In the frozen state, two protein interaction peaks were observed by SANS with corresponding interaction distances at 40 Å as well as 90 Å. On the other hand, both SAXS and SANS data for freeze-dried samples showed three peaks, suggesting interaction distances ranging from ~ 15 Å to 170 Å. Possible interpretations of these interaction peaks will be discussed, as well as the role of sorbitol as a cryoprotectant during the freezing and drying process.

  10. Systematic mutation of bacteriophage T4 lysozyme.

    PubMed

    Rennell, D; Bouvier, S E; Hardy, L W; Poteete, A R

    1991-11-05

    Amber mutations were introduced into every codon (except the initiating AUG) of the bacteriophage T4 lysozyme gene. The amber alleles were introduced into a bacteriophage P22 hybrid, called P22 e416, in which the normal P22 lysozyme gene is replaced by its T4 homologue, and which consequently depends upon T4 lysozyme for its ability to form a plaque. The resulting amber mutants were tested for plaque formation on amber suppressor strains of Salmonella typhimurium. Experiments with other hybrid phages engineered to produce different amounts of wild-type T4 lysozyme have shown that, to score as deleterious, a mutation must reduce lysozyme activity to less than 3% of that produced by wild-type P22 e416. Plating the collection of amber mutants covering 163 of the 164 codons of T4 lysozyme, on 13 suppressor strains that each insert a different amino acid substitutions at every position in the protein (except the first). Of the resulting 2015 single amino acid substitutions in T4 lysozyme, 328 were found to be sufficiently deleterious to inhibit plaque formation. More than half (55%) of the positions in the protein tolerated all substitutions examined. Among (N-terminal) amber fragments, only those of 161 or more residues are active. The effects of many of the deleterious substitutions are interpretable in light of the known structure of T4 lysozyme. Residues in the molecule that are refractory to replacements generally have solvent-inaccessible side-chains; the catalytic Glu11 and Asp20 residues are notable exceptions. Especially sensitive sites include residues involved in buried salt bridges near the catalytic site (Asp10, Arg145 and Arg148) and a few others that may have critical structural roles (Gly30, Trp138 and Tyr161).

  11. Chemical modification of lysozyme, glucose 6-phosphate dehydrogenase, and bovine eye lens proteins induced by peroxyl radicals: role of oxidizable amino acid residues.

    PubMed

    Arenas, Andrea; López-Alarcón, Camilo; Kogan, Marcelo; Lissi, Eduardo; Davies, Michael J; Silva, Eduardo

    2013-01-18

    Chemical and structural alterations to lysozyme (LYSO), glucose 6-phosphate dehydrogenase (G6PD), and bovine eye lens proteins (BLP) promoted by peroxyl radicals generated by the thermal decomposition of 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH) under aerobic conditions were investigated. SDS-PAGE analysis of the AAPH-treated proteins revealed the occurrence of protein aggregation, cross-linking, and fragmentation; BLP, which are naturally organized in globular assemblies, were the most affected proteins. Transmission electron microscopy (TEM) analysis of BLP shows the formation of complex protein aggregates after treatment with AAPH. These structural modifications were accompanied by the formation of protein carbonyl groups and protein hydroperoxides. The yield of carbonyls was lower than that for protein hydroperoxide generation and was unrelated to protein fragmentation. The oxidized proteins were also characterized by significant oxidation of Met, Trp, and Tyr (but not other) residues, and low levels of dityrosine. As the dityrosine yield is too low to account for the observed cross-linking, we propose that aggregation is associated with tryptophan oxidation and Trp-derived cross-links. It is also proposed that Trp oxidation products play a fundamental role in nonrandom fragmentation and carbonyl group formation particularly for LYSO and G6PD. These data point to a complex mechanism of peroxyl-radical mediated modification of proteins with monomeric (LYSO), dimeric (G6PD), and multimeric (BLP) structural organization, which not only results in oxidation of protein side chains but also gives rise to radical-mediated protein cross-links and fragmentation, with Trp species being critical intermediates.

  12. Study of the binding between lysozyme and C10-TAB: determination and interpretation of the partial properties of protein and surfactant at infinite dilution.

    PubMed

    Morgado, Jorge; Aquino-Olivos, Marco Antonio; Martínez-Hernández, Ranulfo; Corea, Mónica; Grolier, Jean Pierre E; del Río, José Manuel

    2008-06-01

    This work examines the binding in aqueous solution, through the experimental determination of specific volumes and specific adiabatic compressibility coefficients, of decyltrimethylammonium bromide to lysozyme and to non-charged polymeric particles (which have been specially synthesized by emulsion polymerization). A method was developed to calculate the specific partial properties at infinite dilution and it was shown that a Gibbs-Duhem type equation holds at this limit for two solutes. With this equation, it is possible to relate the behavior of the partial properties along different binding types at a constant temperature. It was found that the first binding type, specific with high affinity, is related to a significant reduction of surfactant compressibility. The second binding type is accompanied by the unfolding of the protein and the third one is qualitatively identical to the binding of the surfactant to non-charged polymeric particles.

  13. Driving force behind adsorption-induced protein unfolding: a time-resolved X-ray reflectivity study on lysozyme adsorbed at an air/water interface.

    PubMed

    Yano, Yohko F; Uruga, Tomoya; Tanida, Hajime; Toyokawa, Hidenori; Terada, Yasuko; Takagaki, Masafumi; Yamada, Hironari

    2009-01-06

    Time-resolved X-ray reflectivity measurements for lysozyme (LSZ) adsorbed at an air/water interface were performed to study the mechanism of adsorption-induced protein unfolding. The time dependence of the density profile at the air/water interface revealed that the molecular conformation changed significantly during adsorption. Taking into account previous work using Fourier transform infrared (FTIR) spectroscopy, we propose that the LSZ molecules initially adsorbed on the air/water interface have a flat unfolded structure, forming antiparallel beta-sheets as a result of hydrophobic interactions with the gas phase. In contrast, as adsorption continues, a second layer forms in which the molecules have a very loose structure having random coils as a result of hydrophilic interactions with the hydrophilic groups that protrude from the first layer.

  14. Surface protein imprinted core-shell particles for high selective lysozyme recognition prepared by reversible addition-fragmentation chain transfer strategy.

    PubMed

    Li, Qinran; Yang, Kaiguang; Liang, Yu; Jiang, Bo; Liu, Jianxi; Zhang, Lihua; Liang, Zhen; Zhang, Yukui

    2014-12-24

    A novel kind of lysozyme (Lys) surface imprinted core-shell particles was synthesized by reversible addition-fragmentation chain transfer (RAFT) strategy. With controllable polymer shell chain length, such particles showed obviously improved selectivity for protein recognition. After the RAFT initial agent and template protein was absorbed on silica particles, the prepolymerization solution, with methacrylic acid and 2-hydroxyethyl methacrylate as the monomers, and N,N'-methylenebis(acrylamide) as the cross-linker, was mixed with the silica particles, and the polymerization was performed at 40 °C in aqueous phase through the oxidation-reduction initiation. Ater polymerization, with the template protein removal and destroying dithioester groups with hexylamine, the surface Lyz imprinted particles were obtained with controllable polymer chain length. The binding capacity of the Lys imprinted particles could reach 5.6 mg protein/g material, with the imprinting factor (IF) as 3.7, whereas the IF of the control material prepared without RAFT strategy was only 1.6. The absorption equilibrium could be achieved within 60 min. Moreover, Lys could be selectively recognized by the imprinted particles from both a four-proteins mixture and egg white sample. All these results demonstrated that these particles prepared by RAFT strategy are promising to achieve the protein recognition with high selectivity.

  15. DNA, protein binding, cytotoxicity, cellular uptake and antibacterial activities of new palladium(II) complexes of thiosemicarbazone ligands: effects of substitution on biological activity.

    PubMed

    Kalaivani, P; Prabhakaran, R; Dallemer, F; Poornima, P; Vaishnavi, E; Ramachandran, E; Padma, V Vijaya; Renganathan, R; Natarajan, K

    2012-01-01

    The coordination propensities of 4(N,N')-diethylaminosalicylaldehyde-4(N)-substituted thiosemicarbazones (H(2)L(1-4)) were investigated by reacting with an equimolar amount of [PdCl(2)(PPh(3))(2)]. The new complexes were characterized by various spectroscopic techniques. The structure determination of the complexes [Pd(DeaSal-tsc)(PPh(3))] (1), [Pd(DeaSal-mtsc)(PPh(3))] (2) and [Pd(DeaSal-etsc)(PPh(3))] (3) by X-ray crystallography showed that ligands are coordinated in a dibasic tridentate ONS donor fashion forming stable five and six membered chelate rings. The binding ability of complexes (1-4) to calf-thymus DNA (CT DNA) has been explored by absorption and emission titration methods. Based on the observations, an electrostatic and an intercalative binding mode have been proposed. The protein binding studies have been monitored by quenching of tryptophan and tyrosine residues in the presence of complexes using lysozyme as a model protein. As determined by MTT assays, complex 3 exhibited a higher cytotoxic effect towards human lung cancer cell line (A549) and liver cancer cells (HepG2). LDH, NO assay and cellular uptake of the complexes have been studied. Further, antibacterial activity studies of the complexes have been screened against the pathogenic bacteria such as Enterococcus faecalis, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa, MIC50 values of the complexes showed that the complexes exhibited significant activity against the pathogens and among the complexes, 3 exhibited higher activity.

  16. The Effect of Complex Solvents on the Structure and Dynamics of Protein Solutions: the case of Lysozyme in Trehalose/Water Mixtures

    SciTech Connect

    Ghattyvenkatakrishna, Pavan K; Carri, Gustavo A.

    2013-01-01

    We present a Molecular Dynamics simulation study of the effect of trehalose concentration on the structure and dynamics of individual proteins immersed in trehalose/water mixtures. Hen Egg White Lysozyme is used in this study and trehalose concentrations of 0%, 10%, 20%, 30% and 100% by weight are explored. Surprisingly, we have found that changes in trehalose concentration do not change the global structural characteristics of the protein as measured by standard quantities like the mean square deviation, radius of gyration, solvent accessible surface area, inertia tensor and asphericity. Only in the limit of pure trehalose these metrics change significantly. Specifically, we found that the protein is compressed by 2% when immersed in pure trehalose. At the amino acid level there is noticeable rearrangement of the surface residues due to the change in polarity of the surrounding environment with the addition of trehalose. From a dynamic perspective, our computation of the Incoherent Intermediate Scattering Function shows that the protein slows down with increasing trehalose concentration; however, this slowdown is not monotonic. Finally, we also report in-depth results for the hydration layer around the protein including its structure, hydrogen- bonding characteristics and dynamic behavior at different length scales.

  17. Fluorescence Studies of Lysozyme Nucleation

    NASA Technical Reports Server (NTRS)

    Pusey, Marc L.; Smith, Lori

    1998-01-01

    Fluorescence is one of the most powerful tools available for the study of macromolecules. For example, fluorescence can be used to study self association through methods such as anisotropy (the rotational rate of the molecule in solution), quenching (the accessibility of a bound probe to the bulk solution), and resonance energy transfer (measurement of the distance between two species). Fluorescence can also be used to study the local environment of the probe molecules, and the changes in that environment which accompany crystal nucleation and growth. However fluorescent techniques have been very much underutilized in macromolecular growth studies. One major advantage is that the fluorescent species generally must be at low concentration, typically ca 10-5 to 10-6 M. Thus one can study a very wide range of solution conditions, ranging from very high to very low protein concentration, he latter of which are not readily accessible to scattering techniques. We have prepared a number of fluorescent derivatives of chicken egg white lysozyme (CEWL). Fluorescent probes have been attached to two different sites, ASP 101 and the N-terrninal amine, with a sought for use in different lines of study. Preliminary resonance energy transfer studies have been -carried out using pyrene acetic acid (Ex 340 mn, Em 376 nm) lysozyme as a donor and cascade blue (Ex 377 run, Em 423 nm) labeled lysozyme as an acceptor. The emission of both the pyrene and cascade blue probes was followed as a function of the salt protein concentrations. The data show an increase in cascade blue and a concomitant decrease in the pyrene fluorescence as either the salt or protein concentrations are increased, suggesting that the two species are approaching each other close enough for resonance energy transfer to occur. This data can be analyzed to measure the distance between the probe molecules and, knowing their locations on the protein molecule their distances from and orientations with respect to each

  18. Dose-dependent effect of lysozyme upon Candida albicans biofilm

    PubMed Central

    Sebaa, Sarra; Hizette, Nicolas; Boucherit-Otmani, Zahia; Courtois, Philippe

    2017-01-01

    The present study investigated the in vitro effect of lysozyme (0–1,000 µg/ml) on Candida albicans (C. albicans) biofilm development. Investigations were conducted on C. albicans ATCC 10231 and on 10 clinical isolates from dentures. Strains were cultured aerobically at 37°C in Sabouraud broth. Yeast growth was evaluated by turbidimetry. Biofilm biomass was quantified on a polystyrene support by crystal violet staining and on acrylic surfaces by counts of colony forming units. Lysozyme affected biofilm formation to a greater extent than it affected growth. For the ATCC 10231 reference strain, lysozyme acted as a biofilm promotor on polystyrene at the highest concentration tested (1,000 µg/ml, non-physiological). When the reference strain was investigated on acrylic resin support, lysozyme acted as a significant biofilm promotor on rough resin, but less on smooth resin. The attached biomass in the presence of physiological concentrations of lysozyme (10–30 µg/ml) was significantly decreased compared with the hypothetical value of 100% using a one-sample t-test, but a comparison between the different lysozyme conditions using analysis of variance and post hoc tests did not reveal significant differences. In 10 wild strains, different patterns of biofilm formation on polystyrene were observed in the presence of lysozyme. Some strains, characterized by large amounts of biofilm formation in the presence of 1,000 µg/ml lysozyme, were poor biofilm producers at low concentrations of lysozyme. In contrast, some strains that were poor biofilm producers with a high lysozyme concentration were more inhibited by low concentrations of lysozyme. The present study emphasizes the need to develop strategies for biofilm control based on in vitro experiments, and to implement these in clinical trials prior to approval of hygiene products enriched with exocrine proteins, such as lysozyme. Further studies will extend these investigations to other Candida species, and to fungi

  19. Antibacterial peptides and mitochondrial presequences affect mitochondrial coupling, respiration and protein import.

    PubMed

    Hugosson, M; Andreu, D; Boman, H G; Glaser, E

    1994-08-01

    Cecropins A and P1, antibacterial peptides from insects and from pig and some related peptides released respiratory control, inhibited protein import and at higher concentrations also inhibited respiration. However, PR-39, an antibacterial peptide from pig intestine, was found to be almost inert towards mitochondria. The concentrations at which the three mitochondrial functions were effected varied for different peptides. Melittin, magainin and Cecropin-A-(1,13)-Melittin(1,13)-NH2, a hybrid between cecropin A and melittin, were most potent, while the two cecropins acted at higher concentrations. The biosynthesis of cecropin A is known and the intermediates are synthesized. We have used four peptides from this pathway to investigate their effects on coupling, respiration and protein import into mitochondria. Mature cecropin A followed by the preproprotein were most aggressive whereas the intermediates were less active or inert. The efficiency of different derivatives of cecropin A as uncouplers correlates well with their capacity to release membrane potential measured as fluorescence quenching of Rhodamine 123. Inhibition of respiration was found to be dependent on membrane potential and was most pronounced with mature cecropin A, less so with its three precursors. We also found that three peptides derived from mitochondrial presequences showed antibacterial activity. It is concluded that, there are similarities in the functions of antibacterial peptides and mitochondrial presequences, uncoupling activity in mitochondria cannot be correlated with the antibacterial activity (contrary to a previous suggestion), the processing of preprocecropin A may have evolved in such a way that there is a minimum of membrane damage from the intermediates in the pathway, and peptides produced for delivery outside of an animal have evolved to be more aggressive against mitochondria than peptides for delivery inside of the animal.

  20. The boundary molecules in a lysozyme pattern exhibit preferential antibody binding.

    PubMed

    Gao, Pei; Cai, Yuguang

    2008-09-16

    Lysozyme was immobilized on a prefabricated carboxylic acid terminated chemical template, forming a tightly packed, one monolayer thick lysozyme pattern. Polyclonal anti-lysozyme antibodies can bind to the immobilized lysozyme pattern. Atomic force microscope (AFM) observation reveals that the antibodies bind to the lysozyme molecules on the pattern edge before they bind to the lysozyme molecules in the pattern interior. Better spatial accessibility and flexibility of the lysozyme molecules on the pattern edge are used to explain the observed antibody binding preference. The topographies of the lysozyme pattern also affect the antibody binding. The antibodies bind to the edge lysozyme from the top if the lysozyme pattern is half-buried in a 10 A deep channel, whereas the antibodies bind to the edge lysozyme from the side if the lysozyme pattern is immobilized on a protruding terrace. The observed "edge effect" suggests that, for the same protein coverage, reducing the protein pattern feature to the nanoscale will improve the overall binding activity of the immobilized protein toward the antibody.

  1. Isolation and Purification of an Antibacterial Protein from Immune Induced Haemolymph of American Cockroach, Periplaneta americana

    PubMed Central

    Basseri, Hamid Reza; Dadi-Khoeni, Amir; Bakhtiari, Ronak; Abolhassani, Mandan; Hajihosseini-Baghdadabadi, Reza

    2016-01-01

    Background: Antimicrobial peptides play a role as effectors substances in the immunity of vertebrate and invertebrate hosts. In the current study, antimicrobial peptide was isolated from the haemolymph of the American cockroach, Periplaneta americana. Methods: Micrococcus luteus as Gram-positive bacteria and Escherichia coli as Gram-negative bacteria were candidate for injection. Induction was done by injecting both bacteria into the abdominal cavity of two groups of cockroaches separately. The haemolymphs were collected 24 hours after post injection and initially tested against both bacteria. Subsequently, the immune induced haemolymph was purified by high performance liquid chromatography (HPLC) to separate the proteins responsible for the antibacterial activity. Results: The non-induced haemolymph did not show any activity against both bacteria whereas induced haemolymph exhibited high activity against M. luteus but did less against E. coli. Two fractions showed antibacterial activity against M. luteus. Finally the molecular weight of the isolated antibacterial proteins were determined as 72 kDa and 62 kDa using SDS-PAGE. Conclusion: Induced haemolymph of American cockroaches has the ability to produce peptides to combat against Gram-positive bacteria when an immune challenge is mounted. Further work has to be done to sequence of the protein, which it would be advantageous. PMID:28032104

  2. Removal of Protein Capping Enhances the Antibacterial Efficiency of Biosynthesized Silver Nanoparticles

    PubMed Central

    Jain, Navin; Bhargava, Arpit; Rathi, Mohit; Dilip, R. Venkataramana; Panwar, Jitendra

    2015-01-01

    The present study demonstrates an economical and environmental affable approach for the synthesis of “protein-capped” silver nanoparticles in aqueous solvent system. A variety of standard techniques viz. UV-visible spectroscopy, transmission electron microscopy (TEM), energy dispersive spectroscopy (EDS) and X-ray diffraction (XRD) measurements were employed to characterize the shape, size and composition of nanoparticles. The synthesized nanoparticles were found to be homogenous, spherical, mono-dispersed and covered with multi-layered protein shell. In order to prepare bare silver nanoparticles, the protein shell was removed from biogenic nanoparticles as confirmed by UV-visible spectroscopy, FTIR and photoluminescence analysis. Subsequently, the antibacterial efficacy of protein-capped and bare silver nanoparticles was compared by bacterial growth rate and minimum inhibitory concentration assay. The results revealed that bare nanoparticles were more effective as compared to the protein-capped silver nanoparticles with varying antibacterial potential against the tested Gram positive and negative bacterial species. Mechanistic studies based on ROS generation and membrane damage suggested that protein-capped and bare silver nanoparticles demonstrate distinct mode of action. These findings were strengthened by the TEM imaging along with silver ion release measurements using inductively coupled plasma atomic emission spectroscopy (ICP-AES). In conclusion, our results illustrate that presence of protein shell on silver nanoparticles can decrease their bactericidal effects. These findings open new avenues for surface modifications of nanoparticles to modulate and enhance their functional properties. PMID:26226385

  3. An improved 96-well turbidity assay for T4 lysozyme activity

    PubMed Central

    Toro, Tasha B.; Nguyen, Thao P.; Watt, Terry J.

    2015-01-01

    T4 lysozyme (T4L) is an important model system for investigating the relationship between protein structure and function. Despite being extensively studied, a reliable, quantitative activity assay for T4L has not been developed. Here, we present an improved T4L turbidity assay as well as an affinity-based T4L expression and purification protocol. This assay is designed for 96-well format and utilizes conditions amenable for both T4L and other lysozymes. This protocol enables easy, efficient, and quantitative characterization of T4L variants and allows comparison between different lysozymes. Our method: • Is applicable for all lysozymes, with enhanced sensitivity for T4 lysozyme compared to other 96-well plate turbidity assays; • Utilizes standardized conditions for comparing T4 lysozyme variants and other lysozymes; and • Incorporates a simplified expression and purification protocol for T4 lysozyme. PMID:26150996

  4. Lysozyme Photochemistry as a Function of Temperature. The Protective Effect of Nanoparticles on Lysozyme Photostability.

    PubMed

    Oliveira Silva, Catarina; Petersen, Steffen B; Pinto Reis, Catarina; Rijo, Patrícia; Molpeceres, Jesús; Vorum, Henrik; Neves-Petersen, Maria Teresa

    2015-01-01

    The presence of aromatic residues and their close spatial proximity to disulphide bridges makes hen egg white lysozyme labile to UV excitation. UVB induced photo-oxidation of tryptophan and tyrosine residues leads to photochemical products, such as, kynurenine, N-formylkynurenine and dityrosine and to the disruption of disulphide bridges in proteins. We here report that lysozyme UV induced photochemistry is modulated by temperature, excitation power, illumination time, excitation wavelength and by the presence of plasmonic quencher surfaces, such as gold, and by the presence of natural fluorescence quenchers, such as hyaluronic acid and oleic acid. We show evidence that the photo-oxidation effects triggered by 295 nm at 20°C are reversible and non-reversible at 10°C, 25°C and 30°C. This paper provides evidence that the 295 nm damage threshold of lysozyme lies between 0.1 μW and 0.3 μW. Protein conformational changes induced by temperature and UV light have been detected upon monitoring changes in the fluorescence emission spectra of lysozyme tryptophan residues and SYPRO® Orange. Lysozyme has been conjugated onto gold nanoparticles, coated with hyaluronic acid and oleic acid (HAOA). Steady state and time resolved fluorescence studies of free and conjugated lysozyme onto HAOA gold nanoparticles reveals that the presence of the polymer decreased the rate of the observed photochemical reactions and induced a preference for short fluorescence decay lifetimes. Size and surface charge of the HAOA gold nanoparticles have been determined by dynamic light scattering and zeta potential measurements. TEM analysis of the particles confirms the presence of a gold core surrounded by a HAOA matrix. We conclude that HAOA gold nanoparticles may efficiently protect lysozyme from the photochemical effects of UVB light and this nanocarrier could be potentially applied to other proteins with clinical relevance. In addition, this study confirms that the temperature plays a

  5. Lysozyme loading and release from Se doped hydroxyapatite nanoparticles.

    PubMed

    Wang, Yanhua; Hao, Hang; Zhang, Shengmin

    2016-04-01

    Element-substituted hydroxyapatite (HA) based nanocomposites have become a promising therapeutic material for improving bone defect repair. Selenium substituted HA nanoparticles can both induce apoptosis of bone tumor cells and enhance osteointegration. However, the effect of selenite ions on the proteins in combination with the HA nanoparticles remains to be elucidated. Here, we investigated the influence of selenium doping concentration on the loading and release of lysozyme (LSM) as a model protein drug. The selenium substituted HA-LSM composites with different doping concentrations were synthesized and characterized. The subsequent delivery of lysozyme was studied in a phosphate buffer solution (PBS). We found that selenium substituted HA-LSM composites with Se:P=10% showed the highest amount of lysozyme loading (41.7%), whereas the amount of lysozyme loaded in undoped HA nanoparticles was the lowest (34.1%). The doped selenium interacts with lysozyme molecules, which leads to the increase of β-sheet and unordered, and the decrease of self-association, α-helix and β-turns in protein structures. Moreover, selenium addition significantly slows the protein release from HA-LSM composites. The composites with Se:P=10% release lysozyme at the slightly slower rate among the samples with different Se doping concentrations. It also shows that the released lysozyme retains most of its enzymatic activity.

  6. Effect of PEG end-group hydrophobicity on lysozyme interactions in solution characterized by light scattering.

    PubMed

    Priya, M Hamsa; Pratt, L R; Paulaitis, M E

    2011-11-15

    We compare protein-protein and protein-polymer osmotic virial coefficients measured by static light scattering for aqueous solutions of lysozyme with low-molecular-weight, hydroxy-terminated (hPEG) and methyl-terminated (mPEG) poly(ethylene glycol) at two solution conditions: pH 7.0 and 0.01 M ionic strength, and pH 6.2 and 0.8 M ionic strength. We find that adding PEG to aqueous lysozyme solutions makes a net repulsive contribution to lysozyme-lysozyme interactions, independent of ionic strength and PEG end-group hydrophobicity. PEG end-group hydrophobicity has a profound effect on the magnitude of this contribution, however, at low ionic strength where mPEG-lysozyme attractive interactions become significant. The enhanced attractions promote mPEG-lysozyme preferential interactions at the expense of lysozyme self-interactions, which leads to lysozyme-lysozyme interactions that are more repulsive in the presence of mPEG. These preferential interactions also lead to the preferential exclusion of diffusable ions locally around the protein, which results in a pronounced ionic strength dependence of mPEG-mediated lysozyme-lysozyme interactions.

  7. Effects of single-walled carbon nanotubes on lysozyme gelation.

    PubMed

    Tardani, Franco; La Mesa, Camillo

    2014-09-01

    The possibility to disperse carbon nanotubes in biocompatible matrices has got substantial interest from the scientific community. Along this research line, the inclusion of single walled carbon nanotubes in lysozyme-based hydrogels was investigated. Experiments were performed at different nanotube/lysozyme weight ratios. Carbon nanotubes were dispersed in protein solutions, in conditions suitable for thermal gelation. The state of the dispersions was determined before and after thermal treatment. Rheology, dynamic light scattering and different microscopies investigated the effect that carbon nanotubes exert on gelation. The gelation kinetics and changes in gelation temperature were determined. The effect of carbon and lysozyme content on the gel properties was, therefore, determined. At fixed lysozyme content, moderate amounts of carbon nanotubes do not disturb the properties of hydrogel composites. At moderately high volume fractions in carbon nanotubes, the gels become continuous in both lysozyme and nanotubes. This is because percolating networks are presumably formed. Support to the above statements comes by rheology.

  8. Antibacterial and protein-repellent orthodontic cement to combat biofilms and white spot lesions

    PubMed Central

    Zhang, Ning; Chen, Chen; Weir, Michael D.; Bai, Yuxing; Xu, Hockin H. K.

    2016-01-01

    Objectives White spot lesions are the most undesired side-effect of fixed orthodontic treatments. The objectives of this study were to combine nanoparticles of silver (NAg) with 2-methacryloyloxyethyl phosphorylcholine (MPC) to develop a modified resin-modified glass ionomer cement (RMGI) as orthodontic cement with double benefits of antibacterial and protein-repellent capabilities for the first time. Methods NAg and MPC were incorporated into a commercial RMGI. Another commercial orthodontic adhesive also served as control. Enamel shear bond strengths (SBS) were determined. Protein adsorption was measured via a micro bicinchoninic acid method. A dental plaque microcosm biofilm model with human saliva as inoculum was tested. Biofilms adherent on the cement samples and planktonic bacteria in the culture medium away from the cement surfaces were both evaluated for bacterial metabolic activity, colony-forming units (CFU), and lactic acid production. Results Adding 0.1% NAg and 3% MPC to RMGI, and water-aging for 30 days, did not adversely affect the SBS, compared to the unmodified RMGI control (p>0.1). The modified RMGI containing 0.1% NAg and 3% MPC achieved the greatest reduction in protein adsorption, bacterial adhesion, CFU, metabolic activity and lactic acid production. The RMGI containing 0.1% NAg and 3% MPC inhibited not only the bacteria on its surface, but also the bacteria away from the surface in the culture medium. Conclusions The incorporation of double agents (antibacterial NAg + protein-repellent MPC) into RMGI achieved much stronger inhibition of biofilms than using each agent alone. The novel antibacterial and protein-repellent RMGI with substantially-reduced biofilm acids is promising as an orthodontic cement to combat white spot lesions in enamel. PMID:26427311

  9. Adaptive functional diversification of lysozyme in insectivorous bats.

    PubMed

    Liu, Yang; He, Guimei; Xu, Huihui; Han, Xiuqun; Jones, Gareth; Rossiter, Stephen J; Zhang, Shuyi

    2014-11-01

    The role of gene duplication in generating new genes and novel functions is well recognized and is exemplified by the digestion-related protein lysozyme. In ruminants, duplicated chicken-type lysozymes facilitate the degradation of symbiotic bacteria in the foregut. Chicken-type lysozyme has also been reported to show chitinase-like activity, yet no study has examined the molecular evolution of lysozymes in species that specialize on eating insects. Insectivorous bats number over 900 species, and lysozyme expression in the mouths of some of these species is associated with the ingestion of insect cuticle, suggesting a chitinase role. Here, we show that chicken-type lysozyme has undergone multiple duplication events in a major family of insect-eating bats (Vespertilionidae) and that new duplicates have undergone molecular adaptation. Examination of duplicates from two insectivorous bats-Pipistrellus abramus and Scotophilus kuhlii-indicated that the new copy was highly expressed in the tongue, whereas the other one was less tissue-specific. Functional assays applied to pipistrelle lysozymes confirmed that, of the two copies, the tongue duplicate was more efficient at breaking down glycol chitin, a chitin derivative. These results suggest that the evolution of lysozymes in vespertilionid bats has likely been driven in part by natural selection for insectivory.

  10. Monolithic molecularly imprinted cryogel for lysozyme recognition.

    PubMed

    Rabieizadeh, Mohammadmahdi; Kashefimofrad, Seyed Mohammadreza; Naeimpoor, Fereshteh

    2014-10-01

    The application of molecularly imprinted polymers in the selective adsorption of macromolecules such as proteins by monolithic protein-imprinted columns requires a macroporous structure, which can be provided by cryogelation at low temperature in which the formation of ice crystals gives a porous structure to the molecularly imprinted polymer. In this study, we applied this technique to synthesize lysozyme-imprinted polyacrylamide cryogels containing 8% w/v of total monomers and 0.3% w/v of lysozyme. The synthesized cryogel was sponge-like and elastic with very fast swelling and reshaping properties, showing a swelling ratio of 24.5 ± 3 and gel fraction yield of about 72%. It showed an imprinting effect of 1.58 and a separation factor of 1.37 for cytochrome c as the competing protein. Adsorption studies on the cryogel revealed that it follows the Langmuir isotherm, with a maximum theoretical adsorption capacity of 36.3 mg lysozyme per gram of cryogel. Additionally, it was shown that a salt-free rebinding solution at low flow rate and pH = 7.0 is favorable for lysozyme rebinding. This kind of monolithic column promises a wide range of application in separation of various biomolecules due to its preparation simplicity, good rebinding characteristics, and macroporosity.

  11. Cryoelectron microscopy analysis of small heat shock protein 16.5 (Hsp16.5) complexes with T4 lysozyme reveals the structural basis of multimode binding.

    PubMed

    Shi, Jian; Koteiche, Hanane A; McDonald, Ezelle T; Fox, Tara L; Stewart, Phoebe L; McHaourab, Hassane S

    2013-02-15

    Small heat shock proteins (sHSPs) are ubiquitous chaperones that bind and sequester non-native proteins preventing their aggregation. Despite extensive studies of sHSPs chaperone activity, the location of the bound substrate within the sHSP oligomer has not been determined. In this paper, we used cryoelectron microscopy (cryoEM) to visualize destabilized mutants of T4 lysozyme (T4L) bound to engineered variants of the small heat shock protein Hsp16.5. In contrast to wild type Hsp16.5, binding of T4L to these variants does not induce oligomer heterogeneity enabling cryoEM analysis of the complexes. CryoEM image reconstruction reveals the sequestration of T4L in the interior of the Hsp16.5 oligomer primarily interacting with the buried N-terminal domain but also tethered by contacts with the α-crystallin domain shell. Analysis of Hsp16.5-WT/T4L complexes uncovers oligomer expansion as a requirement for high affinity binding. In contrast, a low affinity mode of binding is found to involve T4L binding on the outer surface of the oligomer bridging the formation of large complexes of Hsp16.5. These mechanistic principles were validated by cryoEM analysis of an expanded variant of Hsp16.5 in complex with T4L and Hsp16.5-R107G, which is equivalent to a mutant of human αB-crystallin linked to cardiomyopathy. In both cases, high affinity binding is found to involve conformational changes in the N-terminal region consistent with a central role of this region in substrate recognition.

  12. Purification and characterization of an antibacterial protein from dried fruiting bodies of the wild mushroom Clitocybe sinopica.

    PubMed

    Zheng, Suyue; Liu, Qinghong; Zhang, Guoqing; Wang, Hexiang; Ng, Tzi Bun

    2010-01-01

    A novel antibacterial protein with a molecular mass of 44 kDa has been isolated from dried fruiting bodies of the wild mushroom Clitocybe sinopica. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis showed that the protein was composed of two subunits each with a molecular mass of 22 kDa. Its N-terminal amino-acid sequence, SVQATVNGDKML, has not been reported for other antimicrobial proteins. The purification protocol included ion exchange chromatography on DEAE-cellulose, CM-cellulose and Q-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. The antibacterial protein was adsorbed on all three ion exchangers. The antimicrobial activity profile of the protein against tested bacterial and fungal strains disclosed that it possessed potent antibacterial activity against Agrobacterium rhizogenes, A. tumefaciens, A. vitis, Xanthomonas oryzae and X. malvacearum with a minimum inhibitory concentration mostly below 0.6 microM. However, it had no antibacterial activity against Pseudomonas batatae, Erwinia herbicola, Escherichia coli, and Staphylococcus aureus, and no antifungal activity against Setosphaeria turcica, Fusarium oxysporum, Verticillium dahliae, Bipolaris maydis, and B. sativum. The antibacterial antivity against A. tumefaciens was stable after exposure to 20-60 degrees C for 30 min and to pH 4-9 for 1 h.

  13. Purification of lysozyme using ultrafiltration.

    PubMed

    Ghosh, R; Cui, Z F

    2000-04-20

    This article examines the separation of lysozyme from chicken egg white by ultrafiltration with 25 kDa and 50 kDa MWCO polysulfone membranes. The effects of pH, system hydrodynamics, feed concentration, and transmembrane pressure on permeate flux, lysozyme transmission, purification factor, and productivity have been discussed. With both types of membranes, higher permeate flux and lysozyme transmission were observed at higher pH. Higher lysozyme purity was generally obtained with the 25 kDa MWCO membrane. Purity of lysozyme decreased when the feed concentration was increased. With the 50 kDa MWCO membrane permeate flux, productivity and the purity of lysozyme were found to increase with increase in transmembrane pressure. The possibility of using a two-step ultrafiltration process for achieving high productivity along with high purity of lysozyme was also investigated.

  14. Functional assay for T4 lysozyme-engineered G Protein-Coupled Receptors with an ion channel reporter

    PubMed Central

    Niescierowicz, Katarzyna; Caro, Lydia; Cherezov, Vadim; Vivaudou, Michel; Moreau, Christophe J.

    2013-01-01

    Summary: Structural studies of G protein-coupled receptors (GPCRs) extensively use the insertion of globular soluble protein domains in order to facilitate their crystallization. However, when inserted in the third intracellular loop (i3 loop), the soluble protein domain disrupts their coupling to G proteins and impedes the GPCRs functional characterization by standard G protein-based assays. Therefore, activity tests of crystallization-optimized GPCRs are essentially limited to their ligand binding properties using radioligand binding assays. Functional characterization of additional thermostabilizing mutations requires the insertion of similar mutations in the wild-type receptor to allow G protein-activation tests. We demonstrate that Ion Channel-Coupled Receptor technology is a complementary approach for a comprehensive functional characterization of crystallization-optimized GPCRs and potentially of any engineered GPCR. Ligand-induced conformational changes of the GPCRs are translated into electrical signal and detected by simple current recordings, even though binding of G proteins is sterically blocked by the added soluble protein domain. PMID:24268646

  15. Design of Synthetic Polymer Nanoparticles That Facilitate Resolubilization and Refolding of Aggregated Positively Charged Lysozyme.

    PubMed

    Nakamoto, Masahiko; Nonaka, Tadashi; Shea, Kenneth J; Miura, Yoshiko; Hoshino, Yu

    2016-04-06

    Designed polymer hydrogel nanoparticles (NPs) capable of facilitating resolubilization and refolding of an aggregated protein, positively charged lysozyme, are prepared. NPs designed to interact strongly with denatured lysozyme and relatively weakly with native lysozyme, facilitated resolubilization and refolding of aggregated lysozyme. Such NPs could be prepared by copolymerizing optimized combinations and populations of functional monomers. The refolded lysozyme showed native conformation and enzymatic activity. Eleven grams of aggregated protein was refolded by 1 g of NPs. However, NPs having low affinity to denatured lysozyme and NPs having high affinity to both denatured and native lysozyme showed relatively low facilitation activity. Our results suggest a potential strategy for the design of artificial chaperones with high facilitating activity.

  16. Synergistic action of Galleria mellonella anionic peptide 2 and lysozyme against Gram-negative bacteria.

    PubMed

    Zdybicka-Barabas, Agnieszka; Mak, Pawel; Klys, Anna; Skrzypiec, Krzysztof; Mendyk, Ewaryst; Fiołka, Marta J; Cytryńska, Małgorzata

    2012-11-01

    Lysozyme and antimicrobial peptides are key factors of the humoral immune response in insects. In the present work lysozyme and anionic defense peptide (GMAP2) were isolated from the hemolymph of the greater wax moth Galleria mellonella and their antibacterial activity was investigated. Adsorption of G. mellonella lysozyme on the cell surface of Gram-positive and Gram-negative bacteria was demonstrated using immunoblotting with anti-G. mellonella lysozyme antibodies. Lysozyme effectively inhibited the growth of selected Gram-positive bacteria, which was accompanied by serious alterations of the cell surface, as revealed by atomic force microscopy (AFM) imaging. G. mellonella lysozyme used in concentrations found in the hemolymph of naive and immunized larvae, perforated also the Escherichia coli cell membrane and the level of such perforation was considerably increased by GMAP2. GMAP2 used alone did not perforate E. coli cells nor influence lysozyme muramidase activity. However, the peptide induced a decrease in the turgor pressure of the bacterial cell. Moreover, in the samples of bacteria treated with a mixture of lysozyme and GMAP2 the sodium chloride crystals were found, suggesting disturbance of ion transport across the membrane leading to cell disruption. These results clearly indicated the synergistic action of G. mellonella lysozyme and anionic peptide 2 against Gram-negative bacteria. The reported results suggested that, thanks to immune factors constitutively present in hemolymph, G. mellonella larvae are to some extent protected against infection caused by Gram-negative bacteria.

  17. Dynamics of Lysozyme in Trehalose solutions

    NASA Astrophysics Data System (ADS)

    Ghatty, Pavan; Uberbacher, Edward C.

    2008-03-01

    Anhydrobiosis in Tardigrades and Nematodes has been a topic of constant interest and intrigue in the scientific community. An increase in the concentration of Trehalose has been attributed to the ability of some organisms to survive extreme conditions of temperature, pressure and pH. Although there exist many experimental studies attributing this effect to Trehalose, the molecular details governing the interaction between Trehalose and proteins remains unclear. We have conducted a 20ns study of Lysozyme in varying concentrations of Trehalose in water. Strong and weak hydrogen bonds and hydrophobic interactions between water, Trehalose and protein seem to dictate the interactions in the system. We have observed a hydrogen bonded network of Trehalose around the protein entrapping a layer of water between itself and protein. Lysozyme remains in a near-native conformation throughout the simulation giving hints on the ability of Trehalose in preserving the structure of protiens.

  18. Influence of terminal substitution on structural, DNA, protein binding, anticancer and antibacterial activities of palladium(II) complexes containing 3-methoxy salicylaldehyde-4(N) substituted thiosemicarbazones.

    PubMed

    Kalaivani, P; Prabhakaran, R; Ramachandran, E; Dallemer, F; Paramaguru, G; Renganathan, R; Poornima, P; Vijaya Padma, V; Natarajan, K

    2012-02-28

    The variable chelating behavior of 3-methoxysalicylaldehyde-4(N)-substituted thiosemicarbazones was observed in equimolar reactions with [PdCl(2)(PPh(3))(2)]. The new complexes were characterized by various analytical, spectroscopic techniques (mass, (1)H-NMR, absorption, IR). All the new complexes were structurally characterized by single crystal X-ray diffraction. Crystallographic results showed that the ligands H(2)L(1) and H(2)L(4) are coordinated as binegative tridentate ONS donor ligands in the complexes 1 and 4 by forming six and five member rings. However, the ligands H(2)L(2) and H(2)L(3) bound to palladium in 2 and 3 as uninegative bidentate NS donors by forming a five member chelate ring. From this study, it was found that the substitution on terminal 4(N)-nitrogen may have an influence on the chelating ability of thiosemicarbazone. The presence of hydrogen bonding in 2 and 3 might be responsible for preventing the coordination of phenolic oxygen to the metal ion. The interaction of the complexes with calf-thymus DNA (CT-DNA) has been explored by absorption and emission titration methods. Based on the observations, an electrostatic binding mode of DNA has been proposed. The protein binding studies were monitored by quenching of tryptophan and tyrosine residues in the presence of complexes using Lysozyme as model protein. Antibacterial activity studies of the complexes have been screened against pathogenic bacteria such as Enterococcus faecalis, Staphylococcus aureus, Escherichia coli, Klebsiella pneumonia and Pseudomonas aeruginosa. MIC50 values of the complexes showed that they exhibited significant activity against the pathogens and among them, 3 exhibited higher activity. Further, anticancer activity of the complexes on the lung cancer cell line A549 has also been studied.

  19. Molecular dynamics simulations of lysozyme in water/sugar solutions

    NASA Astrophysics Data System (ADS)

    Lerbret, A.; Affouard, F.; Bordat, P.; Hédoux, A.; Guinet, Y.; Descamps, M.

    2008-04-01

    Structural and dynamical properties of the solvent at the protein/solvent interface have been investigated by molecular dynamics simulations of lysozyme in trehalose, maltose and sucrose solutions. Results are discussed in the framework of the bioprotection phenomena. The analysis of the relative concentration of water oxygen atoms around lysozyme suggests that lysozyme is preferentially hydrated. When comparing the three sugars, trehalose is seen more excluded than maltose and sucrose. The preferential exclusion of sugars from the protein surface induces some differences in the behavior of trehalose and maltose, particularly at 50 and 60 wt% concentrations, that are not observed experimentally in binary sugar/mixtures. The dynamical slowing down of the solvent is suggested to mainly arise from the homogeneity of the water/sugar matrices controlled by the percolation of the sugar hydrogen bonds networks. Furthermore, lysozyme strongly increases relaxation times of solvent molecules at the protein/solvent interface.

  20. Mesoscopic coarse-grained simulations of lysozyme adsorption.

    PubMed

    Yu, Gaobo; Liu, Jie; Zhou, Jian

    2014-05-01

    Coarse-grained simulations are adopted to study the adsorption behavior of lysozyme on different (hydrophobic, neutral hydrophilic, zwitterionic, negatively charged, and positively charged) surfaces at the mesoscopic microsecond time scale (1.2 μs). Simulation results indicate the following: (i) the conformation change of lysozyme on the hydrophobic surface is bigger than any other studied surfaces; (ii) the active sites of lysozyme are faced to the hydrophobic surface with a "top end-on" orientation, while they are exposed to the liquid phase on the hydrophilic surface with a "back-on" orientation; (iii) the neutral hydrophilic surface can induce the adsorption of lysozyme, while the nonspecific protein adsorption can be resisted by the zwitterionic surface; (iv) when the solution ionic strength is low, lysozyme can anchor on the negatively charged surface easily but cannot adsorb on the positively charged surface; (v) when the solution ionic strength is high, the positively charged lysozyme can also adsorb on the like-charged surface; (vi) the major positive potential center of lysozyme, especially the residue ARG128, plays a vital role in leading the adsorption of lysozyme on charged surfaces; (vii) when the ionic strength is high, a counterion layer is formed above the positively charged surface, which is the key factor why lysozyme can adsorb on a like-charged surface. The coarse-grained method based on the MARTINI force field for proteins and the BMW water model could provide an efficient way to understand protein interfacial adsorption behavior at a greater length scale and time scale.

  1. Effect of zinc on lysozyme-like activity of the seastar Marthasterias glacialis (Echinodermata, Asteroidea) mucus.

    PubMed

    Stabili, L; Pagliara, P

    2009-03-01

    Lysozyme represents the best characterized enzyme involved in the self-defense from bacteria. In this study we analysed the effects of zinc on the lysozyme-like activity of the seastar Marthasterias glacialis mucus. This activity, detected by measuring the cleared lysis area of dried Micrococcus lysodeikticus cell walls on Petri dishes, was significantly reduced in presence of zinc. The results are discussed in the light of elucidating the possible relationship between environmental contaminants and increased disease susceptibility in seastars due to the decrease of antibacterial protection. The benefits of using the test of lysozyme activity to monitoring environmental pollution are highlighted.

  2. Plant lectin-like antibacterial proteins from phytopathogens Pseudomonas syringae and Xanthomonas citri.

    PubMed

    Ghequire, Maarten G K; Li, Wen; Proost, Paul; Loris, Remy; De Mot, René

    2012-08-01

    The genomes of Pseudomonas syringae pv. syringae 642 and Xanthomonas citri pv. malvacearum LMG 761 each carry a putative homologue of the plant lectin-like bacteriocin (llpA) genes previously identified in the rhizosphere isolate Pseudomonas putida BW11M1 and the biocontrol strain Pseudomonas fluorescens Pf-5. The respective purified recombinant proteins, LlpAPss642 and LlpAXcm761 , display genus-specific antibacterial activity across species boundaries. The inhibitory spectrum of the P. syringae bacteriocin overlaps partially with those of the P. putida and P. fluorescens LlpAs. Notably, Xanthomonas axonopodis pv. citri str. 306 secretes a protein identical to LlpAXcm761 . The functional characterization of LlpA proteins from two different phytopathogenic γ-proteobacterial species expands the lectin-like bacteriocin family beyond the Pseudomonas genus and suggests its involvement in competition among closely related plant-associated bacteria with different lifestyles.

  3. High-resolution diffraction from crystals of a membrane-protein complex: bacterial outer membrane protein OmpC complexed with the antibacterial eukaryotic protein lactoferrin

    SciTech Connect

    Sundara Baalaji, N.; Acharya, K. Ravi; Singh, T. P.; Krishnaswamy, S. E-mail: mkukrishna@rediffmail.com

    2005-08-01

    Crystals of the complex formed between the bacterial membrane protein OmpC and the antibacterial protein lactoferrin suitable for high-resolution structure determination have been obtained. The crystals belong to the hexagonal space group P6, with unit-cell parameters a = b = 116.3, c = 152.4 Å. Crystals of the complex formed between the outer membrane protein OmpC from Escherichia coli and the eukaryotic antibacterial protein lactoferrin from Camelus dromedarius (camel) have been obtained using a detergent environment. Initial data processing suggests that the crystals belong to the hexagonal space group P6, with unit-cell parameters a = b = 116.3, c = 152.4 Å, α = β = 90, γ = 120°. This indicated a Matthews coefficient (V{sub M}) of 3.3 Å{sup 3} Da{sup −1}, corresponding to a possible molecular complex involving four molecules of lactoferrin and two porin trimers in the unit cell (4832 amino acids; 533.8 kDa) with 63% solvent content. A complete set of diffraction data was collected to 3 Å resolution at 100 K. Structure determination by molecular replacement is in progress. Structural study of this first surface-exposed membrane-protein complex with an antibacterial protein will provide insights into the mechanism of action of OmpC as well as lactoferrin.

  4. Purification and identification of an antibacterial protein from the symbiotic bacteria associated with novel entomopathogenic nematode, Rhabditis (Oscheius) sp.

    PubMed

    Anju, K M; Archana, M M; Mohandas, C; Nambisan, Bala

    2015-04-01

    Entomopathogenic nematodes (EPN) belonging to the families steinernematidae and heterorhabditidae and their symbiotic bacteria Xenorhabdus and Photorhabdus are well-known as biological control agents and are found to produce a wide range of bioactive secondary metabolites. Studies carried out at the Central Tuber Crops Research Institute (CTCRI) on entomopathogenic nematodes resulted in the identification of novel EPN belonging to the family Rhabditidae. This study reports the purification of a high molecular weight antibacterial protein from culture filtrates of a bacterium (Bacillus cereus) symbiotically associated with a novel entomopathogenic nematode Rhabditis (Oscheius) species, maintained at CTCRI laboratory. Fermentation conditions were standardized and optimum antibacterial activity was observed in tryptic soy broth after 48 h incubation at 30 °C. The aqueous extracts yielded antibacterial proteins which were purified by ammonium sulfate precipitation followed by ion exchange chromatography and size exclusion chromatography. Native gel electrophoresis indicated an active protein of molecular mass 220KDa which resolved into a major band of 90 kDa and a minor band of about 40 kDa on SDS-PAGE. The 90 kDa protein showed antibacterial activity and was further analysed by MALDI TOF-MS/MS. The protein was identified as a TQXA (Threonine-glutamine dipeptide) domain containing protein from Bacillus cereus. The protein was found to be active against Bacillus subtilis MTCC2756, Staphylococus aureus MTCC902 and Escherichia coli MTCC 2622 and was thermally stable.

  5. Design of antibacterial biointerfaces by surface modification of poly (ε-caprolactone) with fusion protein containing hydrophobin and PA-1.

    PubMed

    Wang, Xiangxiang; Mao, Jiwei; Chen, Yiming; Song, Dongmin; Gao, Zhendong; Zhang, Xiuming; Bai, Yanling; Saris, Per E J; Feng, Hui; Xu, Haijin; Qiao, Mingqiang

    2017-03-01

    Class IIa bacteriocin pediocin PA-1 has broad-spectrum activity and is a well-characterized candidate food biopreservative. Here, a simple approach is designed to extend the application of pediocin PA-1 in improving the antibacterial activity of electrospun poly(caprolactone) (PCL) grafts through combining PA-1 with HGFI, which is a self-assembled protein with characteristics allowing the modulation of surface properties of other materials originated from Grifola frondosa. Saccharomyces cerevisiae was used as the host for expression of fusion protein PA-1-linker-HGFI (pH) and his-tag purification was used to purify recombinant protein pH. An antibacterial activity assay showed the fusion protein pH retained the biological property of native PA-1. Water contact angle, X-ray photoelectron spectroscopy, immunofluorescence assay and atomic force microscopy indicated the surface properties of HGFI were greatly preserved by the fusion protein pH. Finally, antibacterial activity of pH-modified PCL substrate measurements implied the fusion protein significantly improved the bacterial-resistance of the PCL film through dressing the PCL fibers with the recombinant pH protein. This work presents a new perspective on the application of hydrophobin and pediocin PA-1 in antibacterial medical devices.

  6. Preparation of lysozyme molecularly imprinted polymers and purification of lysozyme from egg white.

    PubMed

    Wang, Xuejiao; Dong, Shaohua; Bai, Quan

    2014-06-01

    Molecular imprinting as a promising and facile separation technique has received much attention because of its high selectivity for target molecules. In this study, lysozyme molecularly imprinted polymers (Lys-MIPs) were successfully prepared by the entrapment method with lysozyme as the template molecule, acrylamide as the functional monomer and N,N-methylenebisacrylamide as the cross-linker. The removal of the template lysozyme from the molecularly imprinted polymers was investigated in detail by two methods. The synthesized Lys-MIPs were characterized by scanning electron microscopy and Fourier transform-infrared, and the adsorption capacity, selectivity and reproducibility of the Lys-MIPs were also evaluated. The maximum adsorption capacity reached 94.8 mg/g, which is twice that of nonmolecularly imprinted polymers, and satisfactory selectivity and reproducibility were achieved. Using the Lys-MIP column, lysozyme could be separated completely from egg white, with purity close to 100% and mass recovery of 98.2%. This illustrated that the synthesized Lys-MIPs had high specific recognition and selectivity to the template lysozyme when they were applied to a mixture of protein standards and a real sample.

  7. Fabrication of Gelatin-Based Electrospun Composite Fibers for Anti-Bacterial Properties and Protein Adsorption.

    PubMed

    Gao, Ya; Wang, Yingbo; Wang, Yimin; Cui, Wenguo

    2016-10-21

    A major goal of biomimetics is the development of chemical compositions and structures that simulate the extracellular matrix. In this study, gelatin-based electrospun composite fibrous membranes were prepared by electrospinning to generate bone scaffold materials. The gelatin-based multicomponent composite fibers were fabricated using co-electrospinning, and the composite fibers of chitosan (CS), gelatin (Gel), hydroxyapatite (HA), and graphene oxide (GO) were successfully fabricated for multi-function characteristics of biomimetic scaffolds. The effect of component concentration on composite fiber morphology, antibacterial properties, and protein adsorption were investigated. Composite fibers exhibited effective antibacterial activity against Staphylococcus aureus and Escherichia coli. The study observed that the composite fibers have higher adsorption capacities of bovine serum albumin (BSA) at pH 5.32-6.00 than at pH 3.90-4.50 or 7.35. The protein adsorption on the surface of the composite fiber increased as the initial BSA concentration increased. The surface of the composite reached adsorption equilibrium at 20 min. These results have specific applications for the development of bone scaffold materials, and broad implications in the field of tissue engineering.

  8. Fabrication of Gelatin-Based Electrospun Composite Fibers for Anti-Bacterial Properties and Protein Adsorption

    PubMed Central

    Gao, Ya; Wang, Yingbo; Wang, Yimin; Cui, Wenguo

    2016-01-01

    A major goal of biomimetics is the development of chemical compositions and structures that simulate the extracellular matrix. In this study, gelatin-based electrospun composite fibrous membranes were prepared by electrospinning to generate bone scaffold materials. The gelatin-based multicomponent composite fibers were fabricated using co-electrospinning, and the composite fibers of chitosan (CS), gelatin (Gel), hydroxyapatite (HA), and graphene oxide (GO) were successfully fabricated for multi-function characteristics of biomimetic scaffolds. The effect of component concentration on composite fiber morphology, antibacterial properties, and protein adsorption were investigated. Composite fibers exhibited effective antibacterial activity against Staphylococcus aureus and Escherichia coli. The study observed that the composite fibers have higher adsorption capacities of bovine serum albumin (BSA) at pH 5.32–6.00 than at pH 3.90–4.50 or 7.35. The protein adsorption on the surface of the composite fiber increased as the initial BSA concentration increased. The surface of the composite reached adsorption equilibrium at 20 min. These results have specific applications for the development of bone scaffold materials, and broad implications in the field of tissue engineering. PMID:27775645

  9. THz characterization of lysozyme at different conformations

    NASA Astrophysics Data System (ADS)

    Globus, Tatiana; Khromova, Tatyana; Lobo, Rebecca; Woolard, Dwight; Swami, Nathan; Fernandez, Erik

    2005-05-01

    This work demonstrates application of Fourier Transform Infrared Spectroscopy (FTIR) technique in the low terahertz frequency range of 10-25 cm-1 to discriminate between different protein conformations and evaluate possible application of THz spectroscopy for monitoring of protein folding-unfolding process. A specific procedure developed earlier for unfolding lysozyme by salt (KSCN) precipitation and refolding the lysozyme molecules by removing of KSCN and dissolving in sodium acetate was used to prepare three different forms of lysozyme. In addition, two standard procedures were used to prepare samples in unfolded conformation: denaturation at high temperature ~95° C followed by fast freezing, and dissolution in 6 M guanidine. Thin, air dried protein films were characterized as well as material in the form of gel. Spectra reveal resonance features in transmission which represent vibrational modes in the protein samples. A great variability of spectral features for the different conformational states showed the sensitivity of vibrational frequencies to the three dimensional structure of proteins. The results obtained on liquid (gel) samples indicate that THz transmission spectroscopy can be used for monitoring folding-unfolding process in a realistic, aqueous environment.

  10. Lysozyme binding onto cat-anionic vesicles.

    PubMed

    Bonincontro, A; Spigone, E; Ruiz Peña, M; Letizia, C; La Mesa, C

    2006-12-15

    Mixing aqueous sodium dodecylsulfate with cetyltrimethylammonium bromide solutions in mole ratios close to (1.7/1.0) allows the formation of cat-anionic vesicles with an excess of negative charges on the outer surface. The vesicular dispersions are mixed with lysozyme, and interact electrostatically with the positive charges on the protein, forming lipo-plexes. Dielectric relaxation, zeta-potential, and light scattering indicate the occurrence of interactions between vesicles and the protein. According to CD, the vesicle-adsorbed protein retains its native conformation. Binding and surface saturation, inferred by dielectric relaxation and zeta-potential, fulfil a charge neutralisation stoichiometry. Adsorbed lysozyme promotes the vesicle clustering and is concomitant with the lipo-plexes flocculation. Above the charge neutralisation threshold, lysozyme in excess remains dispersed in molecular form. Attempts were made to determine in what conditions protein release from the vesicles occurs. Accordingly, the full neutralisation of sodium dodecylsulfate in excess by cetyltrimethylammonium bromide ensures the lipo-plexes break-up, the precipitation of the mixed surfactants and the protein release in native form.

  11. Fabrication of polypyrrole nano-arrays in lysozyme single crystals

    NASA Astrophysics Data System (ADS)

    England, Matt W.; Lambert, Elizabeth M.; Li, Mei; Turyanska, Lyudmila; Patil, Avinash J.; Mann, Stephen

    2012-10-01

    A template-directed method for the synthesis and organization of partially oxidized polypyrrole (PPy) nanoscale arrays within the solvent channels of glutaraldehyde-cross-linked lysozyme single crystals is presented. Macroscopic single crystals of the periodically arranged protein-polymer superstructure are electrically conductive, insoluble in water and organic solvents, and display increased levels of mechanical plasticity compared with native cross-linked lysozyme crystals.A template-directed method for the synthesis and organization of partially oxidized polypyrrole (PPy) nanoscale arrays within the solvent channels of glutaraldehyde-cross-linked lysozyme single crystals is presented. Macroscopic single crystals of the periodically arranged protein-polymer superstructure are electrically conductive, insoluble in water and organic solvents, and display increased levels of mechanical plasticity compared with native cross-linked lysozyme crystals. Electronic supplementary information (ESI) available: Optical microscopy, SEM, TEM images, FTIR spectra and tables, conductivity plot. Experimental methods. See DOI: 10.1039/c2nr32413j

  12. A novel single WAP domain-containing protein isoform with antibacterial relevance in Litopenaeus vannamei.

    PubMed

    Du, Zhi-Qiang; Yuan, Jian-Jun; Ren, Da-Ming

    2015-06-01

    Single WAP domain (SWD)-containing protein is a small protein containing a whey acidic protein (WAP) domain at the C-terminal region. SWD-containing protein exhibits structural similarity to the family of serine proteinase inhibitors. As of this writing, some SWD domain-containing proteins have been identified in crustaceans, and their functions included antibacterial and anti-proteinase activities. We identified a SWD protein isoform gene in Litopenaeus vanname (Lv-SWDi). Very high sequence similarity was found between Lv-SWDi and Lv-SWD. Results of time-course analysis for the gene expression profile showed that Lv-SWDi could produce a rapid feedback and an obvious upregulation at 12 h after Vibrio injection. Endogenous Lv-SWDi protein was obviously upregulated, and the highest expression level was reached at 24 h after Vibrio injection. The purified rLv-SWDi could directly bind to Gram-positive and Gram-negative bacteria. Results of the proteinase inhibitory assay also showed that rLv-SWDi could inhibit secretory protease activity from Bacillus subtilis. Lv-SWDi is a part of an important immunity-relevant gene and may serve important functions in defense against bacteria.

  13. Destroying activity of magnetoferritin on lysozyme amyloid fibrils

    NASA Astrophysics Data System (ADS)

    Kopcansky, Peter; Siposova, Katarina; Melnikova, Lucia; Bednarikova, Zuzana; Timko, Milan; Mitroova, Zuzana; Antosova, Andrea; Garamus, Vasil M.; Petrenko, Viktor I.; Avdeev, Mikhail V.; Gazova, Zuzana

    2015-03-01

    Presence of protein amyloid aggregates (oligomers, protofilaments, fibrils) is associated with many diseases as diabetes mellitus or Alzheimer's disease. The interaction between lysozyme amyloid fibrils and magnetoferritin loaded with different amount of iron atoms (168 or 532 atoms) has been investigated by small-angle X-rays scattering and thioflavin T fluorescence measurements. Results suggest that magnetoferritin caused an iron atom-concentration dependent reduction of lysozyme fibril size.

  14. Lysozyme binds onto functionalized carbon nanotubes.

    PubMed

    Bomboi, Francesca; Tardani, Franco; Gazzoli, Delia; Bonincontro, Adalberto; La Mesa, Camillo

    2013-08-01

    Single walled carbon nanotubes have singular physicochemical properties making them attractive in a wide range of applications. Studies on carbon nanotubes and biological macromolecules exist in literature. However, ad hoc investigations are helpful to better understand the interaction mechanisms. We report on a system consisting of single walled carbon nanotubes and lysozyme. The phenomenology of nanotube-protein interactions and its effects on protein conformation were determined. We investigated the formation of oxidized nanotube-lysozyme conjugates, by studying the effect of both protein concentration and pH. Electrophoretic mobility, dielectric spectroscopy and dynamic light scattering were used to determine the interaction pathways, monitoring the surface charge density and the size of the complexes. The results allowed identifying the conditions of surface saturation at different pH values. The secondary structure of nanotube-adsorbed protein was controlled by circular dichroism; it was observed that it substantially retains its native conformation. Interestingly, we found that the interactions among oxidized nanotubes and lysozyme molecules are mainly of electrostatic nature and easily tunable by varying the pH of the solutions.

  15. Acetylated lysozyme as impurity in lysozyme crystals: constant distribution coefficient

    NASA Astrophysics Data System (ADS)

    Thomas, B. R.; Chernov, A. A.

    2001-11-01

    Hen egg white lysozyme (HEWL) was acetylated to modify molecular charge keeping the molecular size and weight nearly constant. Two derivatives, A and B, more and less acetylated, respectively, were obtained, separated, purified and added to the solution from which crystals of tetragonal HEWL crystals were grown. Amounts of the A and B impurities added were 0.76, 0.38 and 0.1 mg/ml and 0.43, 0.22, 0.1 mg/ml, respectively. The HEWL concentration were 20, 30 and 40 mg/ml. The crystals grown in 18 experiments for each impurity concentration and supersaturation were dissolved and quantities of A or B additives in these crystals were analyzed by cation exchange high performance liquid chromatography. All the data for each set of 18 samples with the different impurity and regular HEWL concentrations is well described by one distribution coefficient K=2.15±0.13 for A and K=3.42±0.25 for B. According to definition of K by Eq. (1) in the text, the condition K=const is equivalent to a decrease of impurity amount in the crystal as the supersaturation increases. The observed independence of the distribution coefficient on both the impurity concentration and supersaturation is explained by the dilution model described in this paper. It shows that the impurity adsorption and incorporation rates are proportional to the impurity concentration and that the growth rate is proportional to the concentration of crystallizing protein in solution. The frequency at which an impurity molecules irreversibly join the crystal was estimated to be 3 s -1, much higher than such frequency for regular crystal molecules 5×10 -2 s -1 at 30 mg/ml lysozyme concentration. Reasons for this inequality are discussed.

  16. Targeted separation of antibacterial peptide from protein hydrolysate of anchovy cooking wastewater by equilibrium dialysis.

    PubMed

    Tang, Wenting; Zhang, Hui; Wang, Li; Qian, Haifeng; Qi, Xiguang

    2015-02-01

    Anchovy (Engraulis japonicus) cooking wastewater (ACWW) is a by-product resulted from the production of boiled-dried anchovies in the seafood processing industry. In this study, the protein hydrolysate of ACWW (ACWWPH) was found to have antimicrobial activity after enzymatic hydrolysis with Protamex. For the targeted screening of antibacterial peptides, liposomes constructed from Staphylococcus aureus membrane lipids were used in an equilibrium dialysis system. The hydrolysate was further purified by liposome equilibrium dialysis combined with high performance liquid chromatography. The purified antimicrobial peptide (ACWWP1) was determined to be GLSRLFTALK, with a molecular weight of 1104.6622Da. The peptide exhibited no haemolytic activity up to a concentration of 512μg/ml. It displayed a dose-dependent bactericidal effect in reconstituted milk. The change in cell surface hydrophobicity and membrane-permeable action of the purified ACWWP1 may have contributed to the antibacterial effect. This study suggests that liposome equilibrium dialysis can be used for the targeted screening of antimicrobial peptides.

  17. Studies on the role of insect hemolymph polypeptides: Galleria mellonella anionic peptide 2 and lysozyme.

    PubMed

    Sowa-Jasiłek, Aneta; Zdybicka-Barabas, Agnieszka; Stączek, Sylwia; Wydrych, Jerzy; Mak, Paweł; Jakubowicz, Teresa; Cytryńska, Małgorzata

    2014-03-01

    The lysozymes are well known antimicrobial polypeptides exhibiting antibacterial and antifungal activities. Their antibacterial potential is related to muramidase activity and non-enzymatic activity resembling the mode of action of cationic defense peptides. However, the mechanisms responsible for fungistatic and/or fungicidal activity of lysozyme are still not clear. In the present study, the anti-Candida albicans activity of Galleria mellonella lysozyme and anionic peptide 2 (AP2), defense factors constitutively present in the hemolymph, was examined. The lysozyme inhibited C. albicans growth in a dose-dependent manner. The decrease in the C. albicans survival rate caused by the lysozyme was accompanied by a considerable reduction of the fungus metabolic activity, as revealed by LIVE/DEAD staining. In contrast, although AP2 reduced C. albicans metabolic activity, it did not influence its survival rate. Our results suggest fungicidal action of G. mellonella lysozyme and fungistatic activity of AP2 toward C. albicans cells. In the presence of AP2, the anti-C. albicans activity of G. mellonella lysozyme increased. Moreover, when the fungus was incubated with both defense factors, true hyphae were observed besides pseudohyphae and yeast-like C. albicans cells. Atomic force microscopy analysis of the cells exposed to the lysozyme and/or AP2 revealed alterations in the cell surface topography and properties in comparison with the control cells. The results indicate synergistic action of G. mellonella AP2 and lysozyme toward C. albicans. The presence of both factors in the hemolymph of naive larvae suggests their important role in the early stages of immune response against fungi in G. mellonella.

  18. Self-assembly of beta-casein and lysozyme.

    PubMed

    Pan, Xiaoyun; Yu, Shaoyong; Yao, Ping; Shao, Zhengzhong

    2007-12-15

    The self-assembly of beta-casein and lysozyme, a linear and a globular protein with isoelectric point of pH 5.0 and 10.7, respectively, was studied. Polydisperse electrostatic complex micelles formed when mixing beta-casein and lysozyme aqueous solutions. After the micelle solution was heated, lysozyme gelated and beta-casein was trapped in the gel, producing narrowly dispersed nanoparticles. The nanoparticles were characterized with laser light scattering, zeta-potential, steady state fluorescence, atomic force microscopy, and transmission electron microscopy. The nanoparticles have spherical shape and their sizes depend on the pH of the heat treatment and the molar ratio of beta-casein to lysozyme. The nanoparticles display amphoteric property and are relatively hydrophobic at pH around 5 and around 10. The net charges on the surface stabilize the nanoparticles in the solution.

  19. Location of Bromide Ions in Tetragonal Lysozyme Crystals

    NASA Technical Reports Server (NTRS)

    Lim, Kap; Nadarajah, Arunan; Forsythe, Elizabeth L.; Pusey, Marc L.

    1998-01-01

    Anions have been shown to play a dominant role in the crystallization of chicken egg white lysozyme from salt solutions. Previous studies employing X-ray crystallography had found one chloride ion binding site in the tetragonal crystal form of the protein and four nitrate ion binding sites in the monoclinic form. In this study the anion positions in the tetragonal form were determined from the difference Fourier map obtained from lysozyme crystal grown in bromide and chloride solutions. Five possible anion binding sites were found in this manner. Some of these sites were in pockets containing basic residues while others were near neutral, but polar, residues. The sole chloride ion binding site found in previous studies was confirmed, while four of these sites corresponded to four binding sites found for nitrate ions in monoclinic crystals. The study suggests that most of the anion binding sites in lysozyme remain unchanged, even when different anions and different crystal forms of lysozyme are employed.

  20. Multiple antibacterial histone H2B proteins are expressed in tissues of American oyster.

    PubMed

    Seo, Jung-Kil; Stephenson, Jeana; Noga, Edward J

    2011-03-01

    We have previously identified a histone H2B isomer (cvH2B-1) from tissue extracts of the bivalve mollusk, the American oyster (Crassostrea virginica). In this paper, we isolate an additional three antibacterial proteins from acidified gill extract by preparative acid-urea-polyacrylamide gel electrophoresis and reversed-phase high performance liquid chromatography. Extraction of these proteins from tissue was best accomplished by briefly boiling the tissues in a weak acetic acid solution. Addition of protease inhibitors while boiling resulted in somewhat lower yields, with one protein being totally absent with this method. Via mass spectrometry, the masses of one of these purified proteins was 13607.0Da (peak 2), which is consistent with the molecular weight of histone H2B. In addition, via western-blotting using anti-calf histone H2B antibody, all three proteins were positive and were thus named cvH2B-2, cvH2B-3 and cvH2B-4. The antibacterial activity of cvH2B-2 was similar to that of cvH2B-1, with activity against a Gram-positive bacterium (Lactococcus lactis subsp. lactis; minimum effective concentration [MEC] 52-57μg/mL) but inactive against Staphylococcus aureus (MEC>250μg/mL). However, both proteins had relatively potent activity against the Gram-negative oyster pathogen Vibrio parahemolyticus (MEC 11.5-14μg/mL) as well as the human pathogen Vibrio vulnificus (MEC 21.3-25.3μg/mL). cvH2B-3 and cvH2B-4 also had similarly strong activity against Vibrio vulnificus. These data provide further evidence for the antimicrobial function of histone H2B isomers in modulating bacterial populations in oyster tissues. The combined estimated concentrations of these histone H2B isomers were far above the inhibitory concentrations for the tested vibrios, including human pathogens. Our results indicate that the highly conserved histone proteins might be important components not only of immune defenses in oysters but have the potential to influence the abundance of a

  1. Fluorescence study of the membrane effects of aggregated lysozyme.

    PubMed

    Kutsenko, Olga K; Trusova, Valeriya M; Gorbenko, Galyna P; Lipovaya, Anna S; Slobozhanina, Ekaterina I; Lukyanenko, Lyudmila M; Deligeorgiev, Todor; Vasilev, Aleksey

    2013-11-01

    The last decade has seen unprecedented upsurge of interest in the structural and toxic properties of particular type of protein aggregates, amyloid fibrils, associated with a number of pathological states. In the present study fluorescence spectroscopy technique has been employed to gain further insight into the membrane-related mechanisms of amyloid toxicity. To this end, erythrocyte model system composed of liposomes and hemoglobin was subjected to the action of oligomeric and fibrillar lysozyme. Acrylamide quenching of lysozyme fluorescence showed that solvent accessibility of Trp62 and Trp108 increases upon the protein fibrillization. Resonance energy transfer measurements suggested the possibility of direct complexation between hemoglobin and aggregated lysozyme. Using the novel squaraine dye SQ-1 it was demonstrated that aggregated lysozyme is capable of inhibiting lipid peroxidation processes. Fluorescent probes pyrene, Prodan and diphenylhexatriene were employed to characterize the membrane-modifying properties of hemoglobin and lysozyme. Both oligomeric and fibrillar forms of lysozyme were found to exert condensing influence on lipid bilayer structure, with the membrane effects of fibrils being less amenable to modulation by hemoglobin.

  2. Chronopotentiometric sensing of specific interactions between lysozyme and the DNA aptamer.

    PubMed

    Ostatná, Veronika; Kasalová-Vargová, Veronika; Kékedy-Nagy, László; Černocká, Hana; Ferapontova, Elena E

    2017-04-01

    Specific DNA-protein interactions are vital for cellular life maintenance processes, such as transcriptional regulation, chromosome maintenance, replication and DNA repair, and their monitoring gives valuable information on molecular-level organization of those processes. Here, we propose a new method of label-free electrochemical sensing of sequence specific binding between the lysozyme protein and a single stranded DNA aptamer specific for lysozyme (DNAapta) that exploits the constant current chronopotentiometric stripping (CPS) analysis at modified mercury electrodes. Specific lysozyme-DNAapta binding was distinguished from nonspecific lysozyme-DNA interactions at thioglycolic acid-modified mercury electrodes, but not at the dithiothreitol-modified or bare mercury electrodes. Stability of the surface-attached lysozyme-DNAapta layer depended on the stripping current (Istr) intensity, suggesting that the integrity of the layer critically depends on the time of its exposure to negative potentials. Stabilities of different lysozyme-DNA complexes at the negatively polarized electrode surface were tested, and it was shown that structural transitions of the specific lysozyme-DNAapta complexes occur in the Istr ranges different from those observed for assemblies of lysozyme with DNA sequences capable of only nonspecific lysozyme-DNA interactions. Thus, the CPS allows distinct discrimination between specific and non-specific protein-DNA binding and provides valuable information on stability of the nucleic acid-protein interactions at the polarized interfaces.

  3. Lysozyme Photochemistry as a Function of Temperature. The Protective Effect of Nanoparticles on Lysozyme Photostability

    PubMed Central

    Oliveira Silva, Catarina; Petersen, Steffen B.; Pinto Reis, Catarina; Rijo, Patrícia; Molpeceres, Jesús; Vorum, Henrik; Neves-Petersen, Maria Teresa

    2015-01-01

    The presence of aromatic residues and their close spatial proximity to disulphide bridges makes hen egg white lysozyme labile to UV excitation. UVB induced photo-oxidation of tryptophan and tyrosine residues leads to photochemical products, such as, kynurenine, N–formylkynurenine and dityrosine and to the disruption of disulphide bridges in proteins. We here report that lysozyme UV induced photochemistry is modulated by temperature, excitation power, illumination time, excitation wavelength and by the presence of plasmonic quencher surfaces, such as gold, and by the presence of natural fluorescence quenchers, such as hyaluronic acid and oleic acid. We show evidence that the photo-oxidation effects triggered by 295 nm at 20°C are reversible and non-reversible at 10°C, 25°C and 30°C. This paper provides evidence that the 295 nm damage threshold of lysozyme lies between 0.1 μW and 0.3 μW. Protein conformational changes induced by temperature and UV light have been detected upon monitoring changes in the fluorescence emission spectra of lysozyme tryptophan residues and SYPRO® Orange. Lysozyme has been conjugated onto gold nanoparticles, coated with hyaluronic acid and oleic acid (HAOA). Steady state and time resolved fluorescence studies of free and conjugated lysozyme onto HAOA gold nanoparticles reveals that the presence of the polymer decreased the rate of the observed photochemical reactions and induced a preference for short fluorescence decay lifetimes. Size and surface charge of the HAOA gold nanoparticles have been determined by dynamic light scattering and zeta potential measurements. TEM analysis of the particles confirms the presence of a gold core surrounded by a HAOA matrix. We conclude that HAOA gold nanoparticles may efficiently protect lysozyme from the photochemical effects of UVB light and this nanocarrier could be potentially applied to other proteins with clinical relevance. In addition, this study confirms that the temperature plays a

  4. Probing lysozyme conformation with light reveals a new folding intermediate.

    PubMed

    Hamill, Andrea C; Wang, Shao-Chun; Lee, C Ted

    2005-11-22

    A means to control lysozyme conformation with light illumination has been developed using the interaction of the protein with a photoresponsive surfactant. Upon exposure to the appropriate wavelength of light, the azobenzene surfactant undergoes a reversible photoisomerization, with the visible-light (trans) form being more hydrophobic than the UV-light (cis) form. As a result, surfactant binding to the protein and, thus, protein unfolding, can be tuned with light. Small-angle neutron scattering (SANS) measurements were used to provide detailed information of the protein conformation in solution. Shape-reconstruction methods applied to the SANS data indicate that under visible light the protein exhibits a native-like form at low surfactant concentrations, a partially swollen form at intermediate concentrations, and a swollen/unfolded form at higher surfactant concentrations. Furthermore, the SANS data combined with FT-IR spectroscopic analysis of the protein secondary structure reveal that unfolding occurs primarily in the alpha domain of lysozyme, while the beta domain remains relatively intact. Thus, the surfactant-unfolded intermediate of lysozyme appears to be a separate structure than the well-known alpha-domain intermediate of lysozyme that contains a folded alpha domain and unfolded beta domain. Because the interactions between the photosurfactant and protein can be tuned with light, illumination with UV light returns the protein to a native-like conformation. Fluorescence emission data of the nonpolar probe Nile red indicate that hydrophobic domains become available for probe partitioning in surfactant-protein solutions under visible light, while the availability of these hydrophobic domains to the probe decrease under UV light. Dynamic light scattering and UV-vis spectroscopic measurements further confirm the shape-reconstruction findings and reveal three discrete conformations of lysozyme. The results clearly demonstrate that visible light causes a

  5. In-vitro antioxidant and antibacterial properties of fermentatively and enzymatically prepared chicken liver protein hydrolysates.

    PubMed

    Chakka, Ashok Kumar; Elias, Mercy; Jini, R; Sakhare, P Z; Bhaskar, N

    2015-12-01

    Protein hydrolysates were prepared from chicken liver using fermentation and enzymatic hydrolysis. The lactic acid bacteria Pediococcus acidilactici NCIM5368 was employed in the fermentation process and a commercial protease (Alcalase® 2.5) was used in enzymatic hydrolysis. Chicken liver hydrolysates prepared by fermentation (FCLH) and enzymatic hydrolysis (ECLH) revealed appreciable amounts of protein [55.85 and 61.34 %; on dry weight basis, respectively]. Fermentation and enzymatic hydrolysis resulted in 14.3 and 26.12 % of degree of hydrolysis. Total antioxidant activity, reducing power, scavenging of superoxide, 2- diphenyl-1-picrylhydrazyl (DPPH) and 2, 2-azino-bis-3-ethyl-benzthiazoline-6-sulphonic acid (ABTS) radicals were determined for both FCLH & ECLH. FCLH & ECLH showed total antioxidant activity of 0.99 and 1.13 μg AAE mg(-1) proteins, respectively; while, they scavenged 96.14 and 92.76 % of DPPH radicals respectively. FCLH showed higher ABTS radical scavenging activity (32.16 %) than ECLH (19.29 %). Superoxide anion scavenging activity of FCLH & ECLH were found to be 95.02 & 88.94 %, respectively. Residues obtained after both treatments also exhibited antioxidant activities. FCLH reported highest antagonistic activity against Listeria monocytogenes (30 mm); while, ECLH showed antibacterial activity only against Micrococcus luteus (12 mm). Both hydrolysates have the potential to be a protein rich ingredient for use in formulated foods and possible help in reduction of oxidative stress.

  6. Tetragonal Lysozyme, From Monomer to Crystal

    NASA Technical Reports Server (NTRS)

    Pusey, Marc L.; Curreri, Peter A. (Technical Monitor)

    2002-01-01

    The data now leads us to a comprehensive model for the process by which tetragonal lysozyme crystals are nucleated and subsequently grow. Lysozyme is typically desolubilized by addition of ionic salts. The salt anions bind to basic and other sites on the protein and promote protein-protein interactions, i.e., initiate the nucleation self assembly process. Formation of protein-protein interactions occurs at the expense of the protein-anion interactions, with the anions being released to the solution. The association follows a defined pattern, forming the "head to side" interactions of the crystal 4(3) helix. The presence of the high salt also promotes hydrophobic interactions between the protein molecules, further tightening their interaction. The solute assembly process persists after crystal nucleation, and the 4(3) helical structures form the subsequent growth units. AFM measurements show that the growth units follow the dimensions of these helices, and that those on the surface are more compact about the c-axis than in the bulk crystal, with adjacent helices riot being in contact. This further supports the role of hydrophobic interactions, as the surface is still in contact with the bulk solution. Once buried within the crystal the protein:salt ratio radically changes and the hydrophobic interactions relax to those measured crystallographically. Thus the crystal growth process recapitulates the initial stages of the nucleation process, and the two seamlessly merge. Experimental evidence, based upon face growth rate, AFM, and fluorescence energy transfer data, for a postulated model of the nucleation of tetragonal lysozyme crystals and how it transitions into crystal growth will be presented.

  7. Elasticity and Strength of Biomacromolecular Crystals: Lysozyme

    NASA Technical Reports Server (NTRS)

    Holmes, A. M.; Witherow, W. K.; Chen, L. Q.; Chernov, A. A.

    2003-01-01

    The static Young modulus, E = 0.1 to 0.5 GPa, the crystal critical strength (sigma(sub c)) and its ratio to E,sigma(sub c)/E is approximately 10(exp 3), were measured for the first time for non cross-linked lysozyme crystals in solution. By using a triple point bending apparatus, we also demonstrated that the crystals were purely elastic. Softness of protein crystals built of hard macromolecules (26 GPa for lysozyme) is explained by the large size of the macromolecules as compared to the range of intermolecular forces and by the weakness of intermolecular bonds as compared to the peptide bond strength. The relatively large reported dynamic elastic moduli (approximately 8 GPa) from resonance light scattering should come from averaging over the moduli of intracrystalline water and intra- and intermolecular bonding.

  8. Membrane effects of lysozyme amyloid fibrils.

    PubMed

    Kastorna, Anna; Trusova, Valeriya; Gorbenko, Galyna; Kinnunen, Paavo

    2012-04-01

    The influence of mature lysozyme fibrils on the structural and physical properties of model membranes composed of phosphatidylcholine (PC) and its mixtures with cardiolipin (CL) (10 mol%) and cholesterol (Chol) (30 mol%) was studied using fluorescent probes DPH, pyrene, Laurdan and MBA. Analysis of pyrene fluorescence spectra along with the measurements of DPH fluorescence anisotropy revealed that the structure of hydrocarbon chains region of lipid bilayer is not affected by the fibrillar aggregates of lysozyme. In contrast, probing the membrane effects by Laurdan and MBA showed the rise of both the generalized polarization of Laurdan and the MBA fluorescence anisotropy, suggesting that amyloid protein induces reduction of bilayer hydration and increase of lipid packing in the interfacial region of model membranes.

  9. Graphene immobilized enzyme/polyethersulfone mixed matrix membrane: Enhanced antibacterial, permeable and mechanical properties

    NASA Astrophysics Data System (ADS)

    Duan, Linlin; Wang, Yuanming; Zhang, Yatao; Liu, Jindun

    2015-11-01

    Enzyme immobilization has been developed to address lots of issues of free enzyme, such as instability, low activity and difficult to retain. In this study, graphene was used as an ideal carrier for lysozyme immobilization, including graphene oxide (GO) immobilized lysozyme (GO-Ly) and chemically reduced graphene oxide (CRGO) immobilized lysozyme (CRGO-Ly). Herein, lysozyme as a bio-antibacterial agent has excellent antibacterial performance and the products of its catalysis are safety and nontoxic. Then the immobilized lysozyme materials were blended into polyethersulfone (PES) casting solution to prepare PES ultrafiltration membrane via phase inversion method. GO and CRGO were characterized by Fourier transform infrared spectroscopy (FTIR), Ultraviolet-visible spectrum (UV), X-ray diffraction (XRD), and transmission electron microscopy (TEM) and the immobilized lysozyme composites were observed by fluorescent microscopy. The results revealed that GO and CRGO were successfully synthesized and lysozyme was immobilized on their surfaces. The morphology, hydrophilicity, mechanical properties, separation properties and antibacterial activity of the hybrid membranes were characterized in detail. The hydrophilicity, water flux and mechanical strength of the hybrid membranes were significantly enhanced after adding the immobilized lysozyme. In the antibacterial experiment, the hybrid membranes exhibited an effective antibacterial performance against Escherichia coli (E. coli).

  10. Penetration and fusion of phospholipid vesicles by lysozyme

    SciTech Connect

    Kim, J.; Kim, H.

    1989-10-01

    The lysozyme-induced fusion of phosphatidylserine/phosphatidylethanolamine vesicles as studied at a wide range of pH is found to correlate well with the binding of this protein to the vesicles. An identical 6000 molecular weight segment of lysozyme at the N-terminal region is found to be protected from tryptic digestion when initially incubated with vesicles at several pH values. Only this segment is labeled by dansyl chloride, which is partitioned into the bilayer. These results suggest the penetration of one segment of lysozyme into the bilayer. Photoactivated labeling of the membrane-penetrating segment of lysozyme with 3-(trifluoromethyl)-3-(({sup 125}I)iodophenyl)diazirine (({sup 125}I)TID) and subsequent identification of the labeled residues by Edman degradation and gamma-ray counting indicate that four amino acids from the N-terminal are located outside the hydrophobic core of the bilayer. Although treatment of the membrane-embedded segment with aminopeptidase failed to cleave any amino acids from the N-terminal, it appears that a loop of lysozyme segment near the N-terminal penetrates into the bilayer at acidic pH. A helical wheel diagram shows that the labeling is done mainly on one surface of the alpha-helix. The penetration kinetics as studied by time-dependent ({sup 125}I)TID labeling coincide with the fusion kinetics, strongly suggesting that the penetration of the lysozyme segment into the vesicles is the cause of the fusion.

  11. An intrinsically shielded hydrogel for the adsorptive recovery of lysozyme.

    PubMed

    Wang, Lu; Zhang, Rongsheng; Eisenthal, Robert; Hubble, John

    2006-07-01

    The present paper addresses the selective recovery of lysozyme from egg white using CM-dextran (carboxymethyldextran)-based hydrogels containing Cibacron Blue as an affinity ligand and co-immobilized BSA intended to act as a shielding agent to reduce non-specific adsorption. Initial studies using pure lysozyme were conducted that indicated that the adsorption capacity increased with ligand density and that adsorption was well described by a Langmuir-type isotherm. The inclusion of BSA as a putative shielding agent did not decrease the adsorption capacity for lysozyme in single-adsorbate experiments. To assess the effectiveness of the shielding strategy, subsequent experiments were conducted with both defined lysozyme/ovalbumin mixtures and hen's-egg white. From these studies, the optimal operating conditions for lysozyme recovery have been determined. These include: optimal initial egg-white concentration [a 10% (v/v) solution of native egg white in the chosen buffer], affinity-ligand density (1.86 mM) and ligand-to-shielding-agent ratio (4:1). The purity of lysozyme obtained from egg white was improved from 69% with a non-shielded hydrogel to 94% with an intrinsically shielded hydrogel. Finally, the possibility of using a protein, rather than dextran-backbone-based, hydrogel was investigated. It was found that BSA could take the place of CM-dextran as the gel backbone in a simplified synthesis, producing a gel which also proved effective for lysozyme recovery with a 30% lysozyme in egg-white solution purified to approx. 92% in a single adsorption-desorption cycle.

  12. Enterococcus faecalis Constitutes an Unusual Bacterial Model in Lysozyme Resistance▿

    PubMed Central

    Hébert, Laurent; Courtin, Pascal; Torelli, Riccardo; Sanguinetti, Maurizio; Chapot-Chartier, Marie-Pierre; Auffray, Yanick; Benachour, Abdellah

    2007-01-01

    Lysozyme is an important and widespread compound of the host constitutive defense system, and it is assumed that Enterococcus faecalis is one of the few bacteria that are almost completely lysozyme resistant. On the basis of the sequence analysis of the whole genome of E. faecalis V583 strain, we identified two genes that are potentially involved in lysozyme resistance, EF_0783 and EF_1843. Protein products of these two genes share significant homology with Staphylococcus aureus peptidoglycan O-acetyltransferase (OatA) and Streptococcus pneumoniae N-acetylglucosamine deacetylase (PgdA), respectively. In order to determine whether EF_0783 and EF_1843 are involved in lysozyme resistance, we constructed their corresponding mutants and a double mutant. The ΔEF_0783 mutant and ΔEF_0783 ΔEF_1843 double mutant were shown to be more sensitive to lysozyme than the parental E. faecalis JH2-2 strain and ΔEF_1843 mutant were. However, compared to other bacteria, such as Listeria monocytogenes or S. pneumoniae, the tolerance of ΔEF_0783 and ΔEF_0783 ΔEF_1843 mutants towards lysozyme remains very high. Peptidoglycan structure analysis showed that EF_0783 modifies the peptidoglycan by O acetylation of N-acetyl muramic acid, while the EF_1843 deletion has no obvious effect on peptidoglycan structure under the same conditions. Moreover, the EF_0783 and EF_1843 deletions seem to significantly affect the ability of E. faecalis to survive within murine macrophages. In all, while EF_0783 is currently involved in the lysozyme resistance of E. faecalis, peptidoglycan O acetylation and de-N-acetylation are not the main mechanisms conferring high levels of lysozyme resistance to E. faecalis. PMID:17785473

  13. Purification and molecular cloning of cDNA for an inducible antibacterial protein of larvae of a coleopteran insect, Holotrichia diomphalia.

    PubMed

    Lee, S Y; Moon, H J; Kurata, S; Kurama, T; Natori, S; Lee, B L

    1994-01-01

    Injection of Escherichia coli into larvae of the coleopteran Holotrichia diomphalia results in the appearance of antibacterial activity in the hemolymph. An antibacterial protein, named holotricin 2, was purified from larvae of this insect and characterized. A cDNA clone for holotricin 2 was isolated and its complete sequence was determined. This protein was found to inhibit the growth of Gram-negative bacteria and to consist of 72-amino acid residues with no cysteine residues. Its amino acid sequence is similar to that of coleoptericine, an antibacterial protein isolated from larvae of the coleopteran Zophobas atratus.

  14. Hydration of thermally denatured lysozyme studied by sorption calorimetry and differential scanning calorimetry.

    PubMed

    Kocherbitov, Vitaly; Arnebrant, Thomas

    2006-05-25

    We have studied hydration (and dehydration) of thermally denatured hen egg lysozyme using sorption calorimetry. Two different procedures of thermal denaturation of lysozyme were used. In the first procedure the protein was denatured in an aqueous solution at 90 degrees C, in the other procedure a sample that contained 20% of water was denatured at 150 degrees C. The protein denatured at 90 degrees C showed very similar sorption behavior to that of the native protein. The lysozyme samples denatured at 150 degrees C were studied at several temperatures in the range of 25-60 degrees C. In the beginning of sorption, the sorption isotherms of native and denatured lysozyme are almost identical. At higher water contents, however, the denatured lysozyme can absorb a greater amount of water than the native protein due to the larger number of available sorption sites. Desorption experiments did not reveal a pronounced hysteresis in the sorption isotherm of denatured lysozyme (such hysteresis is typical for native lysozyme). Despite the unfolded structure, the denatured lysozyme binds less water than does the native lysozyme in the desorption experiments at water contents up to 34 wt %. Glass transitions in the denatured lysozyme were observed using both differential scanning calorimetry and sorption calorimetry. Partial molar enthalpy of mixing of water in the glassy state is strongly exothermic, which gives rise to a positive temperature dependence of the water activity. The changes of the free energy of the protein induced by the hydration stabilize the denatured form of lysozyme with respect to the native form.

  15. Foam fractionation of binary mixtures of lysozyme and albumin.

    PubMed

    Lockwood, C E; Jay, M; Bummer, P M

    2000-06-01

    A nitrogen gas-based foam fractionation method was employed to separate model proteins, bovine serum albumin (BSA) and hen egg white lysozyme, from each other. Fractionation was characterized by the separation ratio and by recovery of proteins in the retentate as a function of the nominal pore size of the gas dispersion frit and solution conditions (pH and ionic strength). For binary mixtures of the proteins at pH 7.4, and ionic strength (mu) of 0.18 M, the recovery of lysozyme and the separation ratio were both dependent on the frit size employed to generate the foam. At low ionic strength (mu = 0.01 M), separation was only somewhat greater with the small pore size frits, although at values significantly lower than those found for high ionic strength. The diminished separations appear to be due to the only slight changes in recoveries observed for BSA and lysozyme.%Separation ratios of lysozyme from BSA in solutions either of high or low ionic strength were maximal at pH values equal to or less than the isoelectric point (pI) of BSA. Separation ratios were lower when foaming was carried out under low compared with high ionic strength. The recovery of lysozyme was enhanced by foaming from solutions of low pH and high ionic strength. Recoveries of BSA were greatest when the molecule was negatively charged. Electrical interactions between the positively charged lysozyme and negatively charged BSA may explain the diminished separation ratios and enhanced recoveries. Enzyme activity studies of lysozyme remaining in the retentate showed no change from prefoam activity.

  16. The identification and characterization of lysozyme from the hard tick Haemaphysalis longicornis.

    PubMed

    Tanaka, Tetsuya; Kawano, Suguru; Nakao, Sumihiro; Umemiya-Shirafuji, Rika; Rahman, Md Morshedur; Boldbaatar, Damdinsuren; Battur, Banzragch; Liao, Min; Fujisaki, Kozo

    2010-12-01

    A full-length cDNA-encoding lysozyme was obtained from cDNA libraries of salivary glands of the hard tick Haemaphysalis longicornis and designated as HlLysozyme. The HlLysozyme sequence represents an open reading frame for a putative signal peptide and the mature protein composed of 121 amino acids. The calculated molecular weight of the protein is 13.7 kDa, and the theoretical isoelectric point is 9.85. HlLysozyme shares 41-79% amino acid sequence identity with the lysozymes of other organisms. The activity of recombinant HlLysozyme expressed in Escherichia coli was confirmed by a lytic zone assay using lyophilized Micrococcus lysodeikticus. The HlLysozyme activity decreased at 70 °C and was demonstrated at acidic side and neutral in a pH range. Elevated gene expression of HlLysozyme was observed when female ticks were challenged with bacteria, suggesting possible roles of lysozyme as an innate immunity of ticks against microorganisms.

  17. Parallel tempering Monte Carlo simulations of lysozyme orientation on charged surfaces

    NASA Astrophysics Data System (ADS)

    Xie, Yun; Zhou, Jian; Jiang, Shaoyi

    2010-02-01

    In this work, the parallel tempering Monte Carlo (PTMC) algorithm is applied to accurately and efficiently identify the global-minimum-energy orientation of a protein adsorbed on a surface in a single simulation. When applying the PTMC method to simulate lysozyme orientation on charged surfaces, it is found that lysozyme could easily be adsorbed on negatively charged surfaces with "side-on" and "back-on" orientations. When driven by dominant electrostatic interactions, lysozyme tends to be adsorbed on negatively charged surfaces with the side-on orientation for which the active site of lysozyme faces sideways. The side-on orientation agrees well with the experimental results where the adsorbed orientation of lysozyme is determined by electrostatic interactions. As the contribution from van der Waals interactions gradually dominates, the back-on orientation becomes the preferred one. For this orientation, the active site of lysozyme faces outward, which conforms to the experimental results where the orientation of adsorbed lysozyme is co-determined by electrostatic interactions and van der Waals interactions. It is also found that despite of its net positive charge, lysozyme could be adsorbed on positively charged surfaces with both "end-on" and back-on orientations owing to the nonuniform charge distribution over lysozyme surface and the screening effect from ions in solution. The PTMC simulation method provides a way to determine the preferred orientation of proteins on surfaces for biosensor and biomaterial applications.

  18. Engineering Escherichia coli for Soluble Expression and Single Step Purification of Active Human Lysozyme

    PubMed Central

    Lamppa, John W.; Tanyos, Sam A.; Griswold, Karl E.

    2012-01-01

    Genetically engineered variants of human lysozyme represent promising leads in the battle against drug-resistant bacterial pathogens, but early stage development and testing of novel lysozyme variants is constrained by the lack of a robust, scalable and facile expression system. While wild type human lysozyme is reportedly produced at 50 – 80 kg per hectare of land in recombinant rice, this plant-based system is not readily scaled down to bench top production, and it is therefore not suitable for development and characterization of novel lysozyme variants. Here, we describe a novel and efficient expression system capable of producing folded, soluble and functional human lysozyme in E. coli cells. To achieve this goal, we simultaneously co-express multiple protein folding chaperones as well as harness the lysozyme inhibitory protein, Ivy. Our strategy exploits E. coli’s ease of culture, short doubling time, and facile genetics to yield upwards of 30 mg/L of soluble lysozyme in a bioreactor system, a 3000-fold improvement over prior efforts in E. coli. Additionally, molecular interactions between lysozyme and a his-tagged Ivy allows for one-step purification by IMAC chromatography, yielding as much as 21 mg/L of purified enzyme. We anticipate that our expression and purification platform will facilitate further development of engineered lysozymes having utility in disease treatment and other practical applications. PMID:23220215

  19. Effects of Purification on the Crystallization of Lysozyme

    NASA Technical Reports Server (NTRS)

    Ewing, Felecia L.; Forsythe, Elizabeth L.; Van Der Woerd, Mark; Pusey, Marc L.

    1996-01-01

    We have additionally purified a commercial lysozyme preparation by cation exchange chromatography, followed by recrystallization. This material is 99.96% pure with respect to macromolecular impurities. At basic pH, the purified lysozyme gave only tetragonal crystals at 20 C. Protein used directly from the bottle, prepared by dialysis against distilled water, or which did not bind to the cation exchange column had considerably altered crystallization behavior. Lysozyme which did not bind to the cation exchange column was subsequently purified by size exclusion chromatography. This material gave predominately bundles of rod-shaped crystals with some small tetragonal crystals at lower pHs. The origin of the bundled rod habit was postulated to be a thermally dependent tetragonal- orthorhombic change in the protein structure. This was subsequently ruled out on the basis of crystallization behavior and growth rate experiments. This suggests that heterogeneous forms of lysozyme may be responsible. These results demonstrate three classes of impurities: (1) small molecules, which may be removed by dialysis; (2) macromolecules, which are removable by chromatographic techniques; and (3) heterogeneous forms of the protein, which can be removed in this case by cation exchange chromatography. Of these, heterogeneous forms of the lysozyme apparently have the greatest affect on its crystallization behavior.

  20. The Anti-sigma Factor RsiV Is a Bacterial Receptor for Lysozyme: Co-crystal Structure Determination and Demonstration That Binding of Lysozyme to RsiV Is Required for σV Activation

    PubMed Central

    Houtman, Jon C.

    2016-01-01

    σ factors provide RNA polymerase with promoter specificity in bacteria. Some σ factors require activation in order to interact with RNA polymerase and transcribe target genes. The Extra-Cytoplasmic Function (ECF) σ factor, σV, is encoded by several Gram-positive bacteria and is specifically activated by lysozyme. This activation requires the proteolytic destruction of the anti-σ factor RsiV via a process of regulated intramembrane proteolysis (RIP). In many cases proteases that cleave at site-1 are thought to directly sense a signal and initiate the RIP process. We previously suggested binding of lysozyme to RsiV initiated the proteolytic destruction of RsiV and activation of σV. Here we determined the X-ray crystal structure of the RsiV-lysozyme complex at 2.3 Å which revealed that RsiV and lysozyme make extensive contacts. We constructed RsiV mutants with altered abilities to bind lysozyme. We find that mutants that are unable to bind lysozyme block site-1 cleavage of RsiV and σV activation in response to lysozyme. Taken together these data demonstrate that RsiV is a receptor for lysozyme and binding of RsiV to lysozyme is required for σV activation. In addition, the co-structure revealed that RsiV binds to the lysozyme active site pocket. We provide evidence that in addition to acting as a sensor for the presence of lysozyme, RsiV also inhibits lysozyme activity. Thus we have demonstrated that RsiV is a protein with multiple functions. RsiV inhibits σV activity in the absence of lysozyme, RsiV binds lysozyme triggering σV activation and RsiV inhibits the enzymatic activity of lysozyme. PMID:27602573

  1. Lysozyme pattern formation in evaporating droplets

    NASA Astrophysics Data System (ADS)

    Gorr, Heather Meloy

    Liquid droplets containing suspended particles deposited on a solid, flat surface generally form ring-like structures due to the redistribution of solute during evaporation (the "coffee ring effect"). The forms of the deposited patterns depend on complex interactions between solute(s), solvent, and substrate in a rapidly changing, far from equilibrium system. Solute self-organization during evaporation of colloidal sessile droplets has attracted the attention of researchers over the past few decades due to a variety of technological applications. Recently, pattern formation during evaporation of various biofluids has been studied due to potential applications in medical screening and diagnosis. Due to the complexity of 'real' biological fluids and other multicomponent systems, a comprehensive understanding of pattern formation during droplet evaporation of these fluids is lacking. In this PhD dissertation, the morphology of the patterns remaining after evaporation of droplets of a simplified model biological fluid (aqueous lysozyme solutions + NaCl) are examined by atomic force microscopy (AFM) and optical microscopy. Lysozyme is a globular protein found in high concentration, for example, in human tears and saliva. The drop diameters, D, studied range from the micro- to the macro- scale (1 microm -- 2 mm). In this work, the effect of evaporation conditions, solution chemistry, and heat transfer within the droplet on pattern formation is examined. In micro-scale deposits of aqueous lysozyme solutions (1 microm < D < 50 microm), the protein motion and the resulting dried residue morphology are highly influenced by the decreased evaporation time of the drop. The effect of electrolytes on pattern formation is also investigated by adding varying concentrations NaCl to the lysozyme solutions. Finally, a novel pattern recognition program is described and implemented which classifies deposit images by their solution chemistries. The results presented in this Ph

  2. Thermodynamic Exploration of Eosin-Lysozyme Binding: A Physical Chemistry and Biochemistry Laboratory Experiment

    ERIC Educational Resources Information Center

    Huisman, Andrew J.; Hartsell, Lydia R.; Krueger, Brent P.; Pikaart, Michael J.

    2010-01-01

    We developed a modular pair of experiments for use in the undergraduate physical chemistry and biochemistry laboratories. Both experiments examine the thermodynamics of the binding of a small molecule, eosin Y, to the protein lysozyme. The assay for binding is the quenching of lysozyme fluorescence by eosin through resonant energy transfer. In…

  3. Antibacterial Activity of the Contact and Complement Systems Is Blocked by SIC, a Protein Secreted by Streptococcus pyogenes*

    PubMed Central

    Frick, Inga-Maria; Shannon, Oonagh; Åkesson, Per; Mörgelin, Matthias; Collin, Mattias; Schmidtchen, Artur; Björck, Lars

    2011-01-01

    Recent studies have shown that activation of complement and contact systems results in the generation of antibacterial peptides. Streptococcus pyogenes, a major bacterial pathogen in humans, exists in >100 different serotypes due to sequence variation in the surface-associated M protein. Cases of invasive and life-threatening S. pyogenes infections are commonly associated with isolates of the M1 serotype, and in contrast to the large majority of M serotypes, M1 isolates all secrete the SIC protein. Here, we show that SIC interferes with the activation of the contact system and blocks the activity of antibacterial peptides generated through complement and contact activation. This effect promotes the growth of S. pyogenes in human plasma, and in a mouse model of S. pyogenes sepsis, SIC enhances bacterial dissemination, results which help explain the high frequency of severe S. pyogenes infections caused by isolates of the M1 serotype. PMID:21068386

  4. Manipulation of lysozyme phase behavior by additives as function of conformational stability.

    PubMed

    Galm, Lara; Morgenstern, Josefine; Hubbuch, Jürgen

    2015-10-15

    Undesired protein aggregation in general and non-native protein aggregation in particular need to be inhibited during bio-pharmaceutical processing to ensure patient safety and to maintain product activity. In this work the potency of different additives, namely glycerol, PEG 1000, and glycine, to prevent lysozyme aggregation and selectively manipulate lysozyme phase behavior was investigated. The results revealed a strong pH dependency of the additive impact on lysozyme phase behavior, lysozyme solubility, crystal size and morphology. This work aims to link this pH dependent impact to a protein-specific parameter, the conformational stability of lysozyme. At pH 3 the addition of 10% (w/v) glycerol, 10% (w/v) PEG 1000, and 1 M glycine stabilized or destabilized lysozymes' native conformation resulting in a modified size of the crystallization area without influencing lysozyme solubility, crystal size and morphology. Addition of 1 M glycine even promoted non-native aggregation at pH 3 whereas addition of PEG 1000 completely inhibited non-native aggregation. At pH 5 the addition of 10% (w/v) glycerol, 10% (w/v) PEG 1000, and 1 M glycine did not influence lysozymes' native conformation, but strongly influenced the position of the crystallization area, lysozyme solubility, crystal size and morphology. The observed pH dependent impact of the additives could be linked to a differing lysozyme conformational stability in the binary systems without additives at pH 3 and pH 5. However, in any case lysozyme phase behavior could selectively be manipulated by addition of glycerol, PEG 1000 and glycine. Furthermore, at pH 5 crystal size and morphology could selectively be manipulated.

  5. Determination of partial unfolding enthalpy for lysozyme upon interaction with dodecyltrimethylammonium bromide using an extended solvation model.

    PubMed

    Behbehani, G Rezaei; Saboury, A A; Taleshi, E

    2008-01-01

    The interactions of dodecyltrimethylammonium bromides (DTABs) with hen egg lysozyme have been investigated at pH = 7.0 and 27 degrees C in phosphate buffer by isothermal titration calorimetry. DTAB interacts endothermically and activate lysozyme. The endothermicity of the lysozyme-DTAB interaction is in marked contrast to the exothermic interactions between sodium dodecyl sulphate (SDS) and lysozyme which have been attributed to specific binding between the anionic sulphate head groups and cationic amino acid residues. The enthalpies of interaction between the cationic surfactant (DTAB) and lysozyme are dominated by the endothermic unfolding of the native structure followed by an exothermic solvation of the lysozyme-DTAB complex by the addition of extra DTAB. A new direct calorimetric method to follow protein denaturation, and the effect of surfactants on the stability of proteins was introduced. The extended solvation model was used to reproduce the enthalpies of lysozyme-DTAB interaction over the whole range of DTAB concentrations. The solvation parameters recovered from the new equation, attributed to the structural change of lysozyme and its biological activity. At low concentrations of DTAB, the binding is mainly electrostatic, with some simultaneous interaction of the hydrophobic tail with nearby hydrophobic patches on the lysozyme. These initial interactions presumably cause some protein unfolding and expose additional hydrophobic sites. The DTAB-induced denaturation enthalpy of lysozyme is 86.46 +/- 0.02 kJ mol(-1).

  6. Synergistic antibacterial mechanism of the Lactobacillus crispatus surface layer protein and nisin on Staphylococcus saprophyticus.

    PubMed

    Sun, Zhilan; Li, Pengpeng; Liu, Fang; Bian, Huan; Wang, Daoying; Wang, Xiaomeng; Zou, Ye; Sun, Chong; Xu, Weimin

    2017-03-21

    SlpB, a surface layer protein isolated from Lactobacillus crispatus, has the potential to enhance the antimicrobial activity of nisin. Previous research indicated that, when combined with nisin, SlpB acted synergistically to inhibit Staphylococcus saprophyticus growth, thus extending the shelf life of chicken meat. In order to understand how SlpB enhances the antibacterial activity of nisin, electron microscopy, confocal laser scanning microscopy, flow cytometry and transmembrane electrical potential analysis were used to study cell wall organization and cell membrane integrity. No remarkable bacteriolytic effects were observed, indicating that cell death could not be attributed to cell lysis, although SlpB caused dramatic modifications of cell wall, thereby altering cell shape. The combination of SlpB and nisin also induced the release of ATP or UV-absorbing materials, as well as sudden dissipation of the transmembrane electrical potential by compromising membrane integrity. Considering that SlpB led to structural disorganization of the cell wall, and nisin access is enhanced to form a stable pore, cell death is a predictable outcome. SlpB significantly enhanced the effect of nisin at half of the minimum inhibitory concentration, which resulted in cell death by destroying the cell wall and cell membrane, therefore providing a new, feasible approach in food preservation.

  7. Elastic Properties of Lysozyme Confined in Nanoporous Polymer Films

    NASA Astrophysics Data System (ADS)

    Wang, Haoyu; Akcora, Pinar

    Retaining the conformational structure and bioactivity of immobilized proteins is important for biosensor designs and drug delivery systems. It is known that confined media provide a protective environment to the encapsulated proteins and prevent diffusion of the denaturant. In this study, different types of proteins (streptavidin, lysozyme and fibrinogen) were chemically attached into the nanopores of poly(methyl methacrylate) thin films. Heterogeneous flat surfaces with varying cylinder pore sizes (10-50 nm) were used to confine proteins of different sizes and shapes. Stiffness of protein functionalized nanopores was measured in nanoindentation experiments. Our results showed that streptavidin behaved more stiffly when pore dimension changed from micron to nanosize. Further, it was found that lysozyme confined within nanopores showed higher specific bioactivity than proteins on flat surfaces. These results on surface elasticity and protein activity may help in understanding protein interactions with surfaces of different topologies and chemistry.

  8. Performance of the lysozyme for promoting the waste activated sludge biodegradability.

    PubMed

    He, Jun-Guo; Xin, Xiao-Dong; Qiu, Wei; Zhang, Jie; Wen, Zhi-Dan; Tang, Jian

    2014-10-01

    The fresh waste activated sludge (WAS) from a lab-scale sequencing batch reactor was used to determine the performance of the lysozyme for promoting its biodegradability. The results showed that a strict linear relationship presented between the degree of disintegration (DDM) of WAS and the lysozyme incubation time from 0 to 240min (R(2) was 0.992, 0.995 and 0.999 in accordance with the corresponding lysozyme/TS, respectively). Ratio of net SCOD increase augmented significantly by lysozyme digestion for evaluating the sludge biodegradability changes. Moreover, the protein dominated both in the EPS and SMP. In addition, the logarithm of SMP contents in supernatant presented an increasing trend similar with the ascending logarithmic relation with the lysozyme incubation time from 0 to 240min (R(2) was 0.960, 0.959 and 0.947, respectively). The SMP, especially the soluble protein, had an important contribution to the improvement of WAS biodegradability.

  9. A global approach to identify novel broad-spectrum antibacterial targets among proteins of unknown function.

    PubMed

    Zalacain, Magdalena; Biswas, Sanjoy; Ingraham, Karen A; Ambrad, Jennifer; Bryant, Alexander; Chalker, Alison F; Iordanescu, Serban; Fan, Jing; Fan, Frank; Lunsford, R Dwayne; O'Dwyer, Karen; Palmer, Leslie M; So, Chi; Sylvester, Daniel; Volker, Craig; Warren, Patrick; McDevitt, Damien; Brown, James R; Holmes, David J; Burnham, Martin K R

    2003-01-01

    Attempted allelic replacement of 144 Streptococcus pneumoniae open reading frames of previously uncharacterized function led to the identification of 36 genes essential for growth under laboratory conditions. Of these, 14 genes (obg, spoIIIJ2, trmU, yacA, yacM, ydiC, ydiE, yjbN, yneS, yphC, ysxC, ytaG, yloI and yxeH4) were also essential in Staphylococcus aureus and Haemophilus influenzae or Escherichia coli, 2 genes (yrrK and ydiB) were only essential in H. influenzae as well as S. pneumoniae and 8 genes were necessary for growth of S.pneumoniae and S. aureus and did not have a homolog in H. influenzae(murD2, ykqC, ylqF, yqeH, ytgP, yybQ) or were not essential in that organism (yqeL, yhcT). The proteins encoded by these genes could represent good targets for novel antibiotics covering different therapeutic profiles. The putative functions of some of these essential proteins, inferred by bioinformatic analysis, are presented. Four mutants, with deletions of loci not essential for in vitro growth, were found to be severely attenuated in a murine respiratory tract infection model, suggesting that not all targets for antibacterial therapeutics are revealed by simple in vitro essentiality testing. The results of our experiments together with those collated from previously reported studies including Bacillus subtilis, E. coli and Mycoplasma sp. demonstrate that gene conservation amongst bacteria does not necessarily indicate that essentiality in one organism can be extrapolated to others. Moreover, this study demonstrates that different experimental procedures can produce apparently contradictory results.

  10. Reentrant condensation of lysozyme: Implications for studying dynamics of lysozyme in aqueous solutions of lithium chloride

    SciTech Connect

    Mamontov, Eugene; O'Neill, Hugh Michael

    2014-01-01

    Recent studies have outlined the use of eutectic solution of lithium chloride in water to study microscopic dynamics of lysozyme in an aqueous solvent that is remarkably similar to pure water in many respects, yet allows experiments over a wide temperature range without the solvent crystallization. The eutectic point in (H2O)R(LiCl) system corresponds to R 7.3, and it is of interest to investigate whether less concentrated aqueous solutions of LiCl could be employed in low-temperature studies of a solvated protein. We have investigated a range of concentrations of lysozyme and LiCl in aqueous solutions to identify systems that do not show phase separation and avoid solvent crystallization on cooling down. Compared to the lysozyme concentration in solution, the concentration of LiCl in the aqueous solvent plays the major role in determining systems suitable for low-temperature studies. We have observed interesting and rich phase behavior reminiscent of reentrant condensation of proteins.

  11. A novel antimicrobial peptide derived from fish goose type lysozyme disrupts the membrane of Salmonella enterica.

    PubMed

    Kumaresan, Venkatesh; Bhatt, Prasanth; Ganesh, Munuswamy-Ramanujam; Harikrishnan, Ramasamy; Arasu, MariadhasValan; Al-Dhabi, Naif Abdullah; Pasupuleti, Mukesh; Marimuthu, Kasi; Arockiaraj, Jesu

    2015-12-01

    In aquaculture, accumulation of antibiotics resulted in development of resistance among bacterial pathogens. Consequently, it became mandatory to find alternative to synthetic antibiotics. Antimicrobial peptides (AMPs) which are described as evolutionary ancient weapons have been considered as promising alternates in recent years. In this study, a novel antimicrobial peptide had been derived from goose type lysozyme (LyzG) which was identified from the cDNA library of freshwater fish Channa striatus (Cs). The identified lysozyme cDNA contains 585 nucleotides which encodes a protein of 194 amino acids. CsLyzG was closely related to Siniperca chuatsi with 92.8% homology. The depicted protein sequence contained a GEWL domain with conserved GLMQ motif, 7 active residues and 2 catalytic residues. Gene expression analysis revealed that CsLyzG was distributed in major immune organs with highest expression in head kidney. Results of temporal expression analysis after bacterial (Aeromonas hydrophila) and fungal (Aphanomyces invadans) challenges indicated a stimulant-dependent expression pattern of CsLyzG. Two antimicrobial peptides IK12 and TS10 were identified from CsLyzG and synthesized. Antibiogram showed that IK12 was active against Salmonella enterica, a major multi-drug resistant (MDR) bacterial pathogen which produces beta lactamase. The IK12 induced loss of cell viability in the bacterial pathogen. Flow cytometry assay revealed that IK12 disrupt the membrane of S. enterica which is confirmed by scanning electron microscope (SEM) analysis that reveals blebs around the bacterial cell membrane. Conclusively, CsLyzG is a potential innate immune component and the identified antimicrobial peptide has great caliber to be used as an ecofriendly antibacterial substance in aquaculture.

  12. In vitro Effects of Selected Saponins on the Production and Release of Lysozyme Activity of Human Monocytic and Epithelial Cell Lines

    PubMed Central

    Helal, Racha; Melzig, Matthias F.

    2011-01-01

    Lysozyme is one of the most important factors of innate immunity and a unique enzybiotic in that it exerts not only antibacterial activity, but also antiviral, anti-inflammatory, anticancer and immunomodulatory activities. The purpose of the present study was to investigate whether in vitro exposure to saponins can affect the release and production of lysozyme activity in human monocytic cells THP-1, and in human epithelial cells HT-29. Lysozyme activity levels in cell culture fluids were measured using highly sensitive fluorescence-based lysozyme activity assay. Majority of the examined saponins were demonstrated to stimulate significantly the release of lysozyme activity of monocytes and epithelial cells after one hour treatment at non-toxic concentrations. On the contrary, cells treated with saponins for longer periods up to 72 hours showed tendency to decrease in the secretion and production of lysozyme activity. However, these inhibitory effects of saponins observed with long-term treatment periods were mostly associated with toxic effects of saponins to cells. The results suggested positive contribution of some saponins to lysozyme release of monocytes and epithelial cells upon short exposure. Furthermore, demonstrated ability of these saponins to enhance the release of lysozyme activity can present a new mechanism contribute to explaining important biological characteristics of saponins, including the antibacterial, antiviral, anti-inflammatory or immune-stimulating properties. PMID:21773070

  13. Expression of recombinant human lysozyme in transgenic chicken promotes the growth of Bifidobacterium in the intestine and improves postnatal growth of chicken.

    PubMed

    Wang, Hai; Wu, Hongping; Wang, Kejun; Cao, Zhichen; Yu, Kun; Lian, Ling; Lian, Zhengxing

    2016-12-01

    Lysozyme is one kind of antimicrobial proteins and often used as feed additive which can defend against pathogenic bacteria and enhance immune function of animals. In this study, we have injected the lentiviral vector expressing recombinant human lysozyme (rhLZ) gene into the blastoderm of chicken embryo to investigate the effect of recombinant human lysozyme on postnatal intestinal microbiota distribution and growth performance of chicken. Successfully, we generated 194 transgenic chickens identified by Southern blot with a positive transgenic rate of 24%. The average concentration of rhLZ was 29.90 ± 6.50 μg/mL in the egg white. Lysozyme in egg white of transgenic chickens had a significantly higher antibacterial activity than those of non-transgenic chickens by lysoplate assay (P < 0.05). The feces of transgenic and non-transgenic chickens were collected and five types of bacteria (Lactobacillus, Salmonella, Bifidobacterium, Staphylococcus aureus and Escherichia coli) were isolated and cultured to detect the impact of rhLZ on gut microbiota. Among the five bacteria, the number of Bifidobacterium in the intestine of those transgenic was significantly increased (P < 0.05). Moreover, the growth traits of the transgenic and non-transgenic chickens were analyzed. It was found that the 6-week shank length, 6-week weight and 18-week weight of transgenic chickens were significantly increased than that of non-transgenic chickens. The results demonstrated that rhLZ-transgenic chicken could promote the growth of Bifidobacterium in the intestine and improve the postnatal growth of chicken.

  14. Analysis of Monomer Aggregation and Crystal Growth Rates of Lysozyme

    NASA Technical Reports Server (NTRS)

    Nadarajah, Arunan

    1996-01-01

    This project was originally conceived to analyze the extensive data of tetragonal lysozyme crystal growth rates collected at NASA/MSFC by Dr. Marc L. Pusey's research group. At that time the lack of analysis of the growth rates was hindering progress in understanding the growth mechanism of tetragonal lysozyme and other protein crystals. After the project was initiated our initial analysis revealed unexpected complexities in the growth rate behavior. This resulted in an expansion in the scope of the project to include a comprehensive investigation of the growth mechanisms of tetragonal lysozyme crystals. A discussion of this research is included as well a list of presentations and publications resulting from the research. This project contributed significantly toward the education of several students and fostered extensive collaborations between investigators.

  15. The folding-unfolding transition of equine lysozyme

    NASA Astrophysics Data System (ADS)

    Haezebrouck, P.; Van Dael, H.

    1993-03-01

    A detailed study of the chemical and thermal unfolding transition of equine lysozyme in the presence and in the absence of Ca 2+ gives evidence for a two-step unfolding process. The pretransition can be related to the transfer of exposed Trp groups to the protein interior.

  16. Lysozyme net charge and ion binding in concentrated aqueous electrolyte solutions

    SciTech Connect

    Kuehner, Daniel E.; Engmann, Jan; Fergg, Florian; Wernick, Meredith; Blanch, Harvey W.; Prausnitz, John M.

    1999-02-01

    Hydrogen-ion titrations were conducted for hen-egg-white lysozyme in solutions of potassium chloride over the range pH 2.5--11.5 and for ionic strengths to 2.0 M. The dependence of lysozyme`s net proton charge, z{sub p}, on pH and ionic strength in potassium chloride solution is measured. From the ionic-strength dependence of z{sub p}, interactions of lysozyme with potassium and chloride ions are calculated using the molecular-thermodynamic theory of Fraaije and Lyklema. Lysozyme interacts preferentially with up to 12 chloride ions at pH 2.5. The observed dependence of ion-protein interactions on pH and ionic strength is explained in terms of electric-double-layer theory. New experimental pK{sub a} data are reported for 11 amino acids in potassium chloride solutions of ionic strength to 3.0 M.

  17. [Separation and purification of lysozyme from egg white by high performance cation-exchange chromatography].

    PubMed

    Li, Rong; Chen, Guo-liang

    2002-05-01

    A new method used to separate and purify lysozyme from egg white by high performance cation-exchange chromatography has been established. The process conditions for purifying lysozyme were also discussed in detail. The procedure involved that homogenization of the egg white sample, preliminary purification with sodium chloride, and chromatographic separation by the weak cation exchange column (XIDACE-WCX). The experimental results showed that the purified lysozyme and other impurity proteins were completely separated. By using bioactivity assay, the recovery of lysozyme was 107%, and the specific activity was 15,467 U/mg through the column. Its purity was raised 5.6-fold. The collected fraction with activity was detected by size-exclusion chromatography (SEC). The purified lysozyme was homogeneous. Compared with the traditional soft-based low pressure ion-exchange chromatography, the developed method is rapid and effective.

  18. Molecular characterisation, evolution and expression analysis of g-type lysozymes in Ciona intestinalis.

    PubMed

    Di Falco, Felicia; Cammarata, Matteo; Vizzini, Aiti

    2017-02-01

    Lysozyme is an important defense molecule of the innate immune system. Known for its bactericidal properties, lysozyme catalyzes the hydrolysis of b-(1,4)-glycosidic bonds between the N-acetyl glucosamine and N-acetyl muramic acid in the peptidoglycan layer of bacterial cell walls. In this study, the complete coding sequence of four g-type lysozymes were identified in Ciona intestinalis. Phylogenetic analysis and modelling supported the hypothesis of a close relationship with the vertebrate g-type lysozymes suggesting that the C. intestinalis g-type lysozyme genes (CiLys-g1, Cilys-g2, CiLys-g3, CiLys-g4) share a common ancestor in the chordate lineage. Protein motif searches indicated that C. intestinalis g-type lysozymes contain a GEWL domain with a GXXQ signature, typical of goose lysozymes. Quantitative Real-Time PCR analysis results showed that transcripts are expressed in various tissues from C. intestinalis. In order to determine the involvement of C. intestinalis g-type lysozymes in immunity, their expression was analyzed in the pharynx, showing that transcripts were significantly up-regulated in response to a challenge with lipopolysaccharide (LPS). These data support the view that CiLys g-type are molecules with potential for immune defense system against bacterial infection.

  19. Engineered regulation of lysozyme by the SH3-CB1 binding interaction.

    PubMed

    Pham, Elizabeth; Truong, Kevin

    2012-06-01

    The ability to design proteins with desired properties by using protein structural information will allow us to create high-value therapeutic and diagnostic products. Using the protein structures of lambda lysozyme and the SH3 domain of human Crk, we designed a synthetic protein switch that controls the activity of lysozyme by sterically hindering its active cleft through the binding of SH3 to its CB1 peptide-binding partner. First, several fusion protein designs with lysozyme and CB1 were modeled to determine the one with greatest steric effect in the presence of SH3. Next, the selected fusion protein was created and tested in vitro. In the absence of SH3, the lysozyme-CB1 fusion protein functioned normally. In the presence of SH3, the lysozyme activity was inhibited and with the addition of excess CB1 peptides to compete for SH3 binding, the lysozyme activity was restored. Lastly, this structure-based strategy can be used to engineer synthetic regulation by peptide-domain-binding interfaces into a variety of proteins.

  20. Osteopontin That Is Elevated in the Airways during COPD Impairs the Antibacterial Activity of Common Innate Antibiotics.

    PubMed

    Gela, Anele; Bhongir, Ravi K V; Mori, Michiko; Keenan, Paul; Mörgelin, Matthias; Erjefält, Jonas S; Herwald, Heiko; Egesten, Arne; Kasetty, Gopinath

    2016-01-01

    Bacterial infections of the respiratory tract contribute to exacerbations and disease progression in chronic obstructive pulmonary disease (COPD). There is also an increased risk of invasive pneumococcal disease in COPD. The underlying mechanisms are not fully understood but include impaired mucociliary clearance and structural remodeling of the airways. In addition, antimicrobial proteins that are constitutively expressed or induced during inflammatory conditions are an important part of the airway innate host defense. In the present study, we show that osteopontin (OPN), a multifunctional glycoprotein that is highly upregulated in the airways of COPD patients co-localizes with several antimicrobial proteins expressed in the airways. In vitro, OPN bound lactoferrin, secretory leukocyte peptidase inhibitor (SLPI), midkine, human beta defensin-3 (hBD-3), and thymic stromal lymphopoietin (TSLP) but showed low or no affinity for lysozyme and LL-37. Binding of OPN impaired the antibacterial activity against the important bacterial pathogens Streptococcus pneumoniae and Pseudomonas aeruginosa. Interestingly, OPN reduced lysozyme-induced killing of S. pneumoniae, a finding that could be explained by binding of OPN to the bacterial surface, thereby shielding the bacteria. A fragment of OPN generated by elastase of P. aeruginosa retained some inhibitory effect. Some antimicrobial proteins have additional functions. However, the muramidase-activity of lysozyme and the protease inhibitory function of SLPI were not affected by OPN. Taken together, OPN can contribute to the impairment of innate host defense by interfering with the function of antimicrobial proteins, thus increasing the vulnerability to acquire infections during COPD.

  1. Osteopontin That Is Elevated in the Airways during COPD Impairs the Antibacterial Activity of Common Innate Antibiotics

    PubMed Central

    Mori, Michiko; Keenan, Paul; Mörgelin, Matthias; Erjefält, Jonas S.; Herwald, Heiko; Egesten, Arne; Kasetty, Gopinath

    2016-01-01

    Bacterial infections of the respiratory tract contribute to exacerbations and disease progression in chronic obstructive pulmonary disease (COPD). There is also an increased risk of invasive pneumococcal disease in COPD. The underlying mechanisms are not fully understood but include impaired mucociliary clearance and structural remodeling of the airways. In addition, antimicrobial proteins that are constitutively expressed or induced during inflammatory conditions are an important part of the airway innate host defense. In the present study, we show that osteopontin (OPN), a multifunctional glycoprotein that is highly upregulated in the airways of COPD patients co-localizes with several antimicrobial proteins expressed in the airways. In vitro, OPN bound lactoferrin, secretory leukocyte peptidase inhibitor (SLPI), midkine, human beta defensin-3 (hBD-3), and thymic stromal lymphopoietin (TSLP) but showed low or no affinity for lysozyme and LL-37. Binding of OPN impaired the antibacterial activity against the important bacterial pathogens Streptococcus pneumoniae and Pseudomonas aeruginosa. Interestingly, OPN reduced lysozyme-induced killing of S. pneumoniae, a finding that could be explained by binding of OPN to the bacterial surface, thereby shielding the bacteria. A fragment of OPN generated by elastase of P. aeruginosa retained some inhibitory effect. Some antimicrobial proteins have additional functions. However, the muramidase-activity of lysozyme and the protease inhibitory function of SLPI were not affected by OPN. Taken together, OPN can contribute to the impairment of innate host defense by interfering with the function of antimicrobial proteins, thus increasing the vulnerability to acquire infections during COPD. PMID:26731746

  2. Human lung lysozyme: sources and properties.

    PubMed

    Konstan, M W; Chen, P W; Sherman, J M; Thomassen, M J; Wood, R E; Boat, T F

    1981-01-01

    Lysozyme in human airway secretions is thought to defend the lung against airborne bacteria. Although lysozyme has been purified and characterized from human tears, milk, saliva, and other sources (1-5), human lung lysozyme has received little attention except for measurements of concentrations in sputum (6, 7), immunocytochemical and histochemical localization (8-12),and studies of secretion by alveolar macrophages (13). This study was designed to identify the sources of secreted lung lysozyme, to quantitate the secretory activities of the various sources,and to compare the properties of lysozyme from lung cells with those from other tissues.

  3. The refined structures of goose lysozyme and its complex with a bound trisaccharide show that the "goose-type" lysozymes lack a catalytic aspartate residue.

    PubMed

    Weaver, L H; Grütter, M G; Matthews, B W

    1995-01-06

    The structure of goose egg-white lysozyme (GEWL) has been refined to an R-value of 15.9% at 1.6 A resolution. Details of the structure determination, the refinement and the structure itself are presented. The structure of a complex of the enzyme with the trisaccharide of N-acetyl glucosamine has also been determined and refined at 1.6 A resolution. The trisaccharide occupies sites analogous to the B, C and D subsites of chicken (HEWL) and phage T4 (T4L) lysozymes. All three lysozymes (GEWL, HEWL and T4L) display the same characteristic set of bridging hydrogen bonds between backbone atoms of the protein and the 2-acetamido group of the saccharide in subsite C. Glu73 of GEWL is seen to correspond closely to Glu35 of HEWL (and to Glu11 of T4L) and supports the established view that this group is critically involved in the catalytic mechanism. There is, however, no obvious residue in goose lysozyme that is a counterpart of Asp52 of chicken lysozyme (or of Asp20 in T4L), suggesting that a second acidic residue is not essential for the catalytic activity of goose lysozyme, and may not be required for the activity of other lysozymes.

  4. Covalent immobilization of lysozyme on ethylene vinyl alcohol films for nonmigrating antimicrobial packaging applications.

    PubMed

    Muriel-Galet, V; Talbert, J N; Hernandez-Munoz, P; Gavara, R; Goddard, J M

    2013-07-10

    The objective of this study was to develop a new antimicrobial film, in which lysozyme was covalently attached onto two different ethylene vinyl alcohol copolymers (EVOH 29 and EVOH 44). The EVOH surface was modified with UV irradiation treatment to generate carboxylic acid groups, and lysozyme was covalently attached to the functionalized polymer surface. Surface characterization of control and modified films was performed using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and dye assay. The value of protein loading after attachment on the surface was 8.49 μg protein/cm(2) and 5.74 μg protein/cm(2) for EVOH 29 and EVOH 44, respectively, after 10 min UV irradiation and bioconjugation. The efficacy of the EVOH-lysozyme films was assessed using Micrococcus lysodeikticus. The antimicrobial activity of the films was tested against Listeria monocytogenes and was similar to an equivalent amount of free enzyme. The reduction was 1.08 log for EVOH 29-lysozyme, 0.95 log for EVOH 44-lysozyme, and 1.34 log for free lysozyme. This work confirmed the successful use of lysozyme immobilization on the EVOH surface for antimicrobial packaging.

  5. Interaction between lysozyme and procyanidin: multilevel structural nature and effect of carbohydrates.

    PubMed

    Liang, Miao; Liu, Rui; Qi, Wei; Su, Rongxin; Yu, Yanjun; Wang, Libing; He, Zhimin

    2013-06-01

    The interaction of procyanidins with proteins has aroused extensive attention due to its important relationship with the bioavailability and astringent property of polyphenols. In the present work, we have investigated the interactions of lysozyme with procyanidin dimer (B3) using various biophysical approaches, which aims to provide insights into the mechanism of protein/polyphenol aggregation. Procyanidin B3 spontaneously binds lysozyme, inducing the multilevel structural changes in lysozyme and the formation of insoluble complexes. The relationship between lysozyme aggregation and the loss of enzymatic activity was monitored using dynamic light scattering and fluorescence quenching. The influences of two carbohydrates (gum arabic and sucrose) on lysozyme/B3 aggregation were also studied. Gum arabic effectively inhibited the formation of insoluble aggregates, but was unable to restore the fluorescence and activity of lysozyme. However, sucrose concomitantly decreased the aggregate size with the recovery of fluorescence and lysozyme activity. These results proposed two probable mechanisms by which these two carbohydrates inhibit protein/polyphenol aggregation.

  6. Multistate Mechanism of Lysozyme Denaturation through Synchronous Analysis of Raman Spectra.

    PubMed

    Xing, Lei; Lin, Ke; Zhou, Xiaoguo; Liu, Shilin; Luo, Yi

    2016-10-10

    The denaturation mechanism of hen egg lysozyme is still controversial. In this study, Raman spectroscopy was employed to study the thermal and chemical denaturation mechanisms of lysozyme. All of the Raman bands were synchronously recorded and analyzed during the denaturation process. It was found that the Raman bands of the side groups changed before the bands of skeleton groups. This directly reveals the three-state mechanism of thermal denaturation of lysozyme. The preferential change of the side groups was also observed in the chemical denaturation of lysozyme by guanidine hydrochloride. Moreover, it was found that the Raman bands of the groups on the surface of lysozyme changed before those of the other groups. This indicates that the chemical denaturants interact with the protein surface before the protein core in each step and the chemical denaturation of lysozyme conforms to the multistate and outside-in mechanisms. The synchronous Raman study not only reveals the multistate mechanism of lysozyme denaturation but also demonstrates that this synchronous Raman analysis is a powerful method to study the denaturation mechanisms of other proteins.

  7. Synthesis and structure-function study about tenecin 1, an antibacterial protein from larvae of Tenebrio molitor.

    PubMed

    Lee, K H; Hong, S Y; Oh, J E

    1998-11-13

    Tenecin 1, an inducible antibacterial protein secreted in the larvae of Tenebrio molitor, has a long N-terminal loop and common structural feature of insect defensin family corresponding to cysteine stabilized alpha/beta motif. To study the function of the N-terminal loop and disulfide bridges, N-terminal loop deleted tenecin 1, reduced tenecin 1 and tenecin 1 were chemically synthesized and their activities were measured. N-terminal loop deleted tenecin and reduced tenecin 1 did not show antibacterial activity. Circular dichroism (CD) spectroscopy data revealed that the alpha-helical content of tenecin 1 and the other proteins increased in the presence of 50% (v/v) trifluoroethanol (TFE) and the alpha-helical content of tenecin 1 was much higher than that of the other proteins in buffer with or without 50% (v/v) TFE. These results suggest that disulfide bridges are necessary for the activity structure and the N-terminal loop plays an important role in the increase of alpha-helix in the membrane mimetic environment and the activity.

  8. Lysozyme activity of the Ruminococcus champanellensis cellulosome.

    PubMed

    Moraïs, Sarah; Cockburn, Darrell W; Ben-David, Yonit; Koropatkin, Nicole M; Martens, Eric C; Duncan, Sylvia H; Flint, Harry J; Mizrahi, Itzhak; Bayer, Edward A

    2016-12-01

    Ruminococcus champanellensis is a keystone species in the human gut that produces an intricate cellulosome system of various architectures. A variety of cellulosomal enzymes have been identified, which exhibit a range of hydrolytic activities on lignocellulosic substrates. We describe herein a unique R. champanellensis scaffoldin, ScaK, which is expressed during growth on cellobiose and comprises a cohesin module and a family 25 glycoside hydrolase (GH25). The GH25 is non-autolytic and exhibits lysozyme-mediated lytic activity against several bacterial species. Despite the narrow acidic pH curve, the enzyme is active along a temperature range from 2 to 85°C and is stable at very high temperatures for extended incubation periods. The ScaK cohesin was shown to bind selectively to the dockerin of a monovalent scaffoldin (ScaG), thus enabling formation of a cell-free cellulosome, whereby ScaG interacts with a divalent scaffodin (ScaA) that bears the enzymes either directly or through additional monovalent scaffoldins (ScaC and ScaD). The ScaK cohesin also interacts with the dockerin of a protein comprising multiple Fn3 domains that can potentially promote adhesion to carbohydrates and the bacterial cell surface. A cell-free cellulosomal GH25 lysozyme may provide a bacterial strategy to both hydrolyze lignocellulose and repel eventual food competitors and/or cheaters.

  9. Effects of oligomeric lysozyme on structural state of model membranes.

    PubMed

    Gorbenko, Galyna; Trusova, Valeriya

    2011-03-01

    The ability of oligomeric lysozyme to modify the molecular organization of the model bilayer membranes composed of phosphatidylcholine (PC) and its mixtures with phosphatidylglycerol (PG) or cholesterol (Chol) was assessed using fluorescent probes 6-propionyl-2-dimethylaminonaphthalene (Prodan), 4-dimethylaminochalcone (DMC), pyrene and 1,6-diphenyl-1,3,5-hexatriene (DPH). The observed changes in the fluorescence characteristics of polarity-sensitive probes Prodan and DMC, located in interfacial bilayer region, were interpreted due to the partial dehydration of the glycerol backbone, which was under the influence of aggregated protein. Cholesterol was found to prevent the perturbations of membrane polar part by lysozyme aggregates. Analysis of the pyrene excimerization data revealed an oligomer-induced reduction in bilayer free volume, presumably caused by an increased packing density of hydrocarbon chains. This effect proved to be virtually independent of membrane composition. It was demonstrated that membranotropic activity of oligomeric lysozyme markedly exceeds that of monomeric protein. The biological significance of the results obtained is twofold, implicating the general membrane-mediated mechanisms of oligomer toxicity and specific pathways of lysozyme fibrillogenesis in vivo associated with familial nonneuropathic systemic amyloidosis.

  10. Structural characteristics of hydration sites in lysozyme.

    PubMed

    Soda, Kunitsugu; Shimbo, Yudai; Seki, Yasutaka; Taiji, Makoto

    2011-06-01

    A new method is presented for determining the hydration site of proteins, where the effect of structural fluctuations in both protein and hydration water is explicitly considered by using molecular dynamics simulation (MDS). The whole hydration sites (HS) of lysozyme are composed of 195 single HSs and 38 clustered ones (CHS), and divided into 231 external HSs (EHS) and 2 internal ones (IHS). The largest CHSs, 'Hg' and 'Lβ', are the IHSs having 2.54 and 1.35 mean internal hydration waters respectively. The largest EHS, 'Clft', is located in the cleft region. The real hydration structure of a CHS is an ensemble of multiple structures. The transition between two structures occurs through recombinations of some H-bonds. The number of the experimental X-ray crystal waters is nearly the same as that of the estimated MDS hydration waters for 70% of the HSs, but significantly different for the rest of HSs.

  11. Inhibition of the growth of Bacillus subtilis DSM10 by a newly discovered antibacterial protein from the soil metagenome

    PubMed Central

    O’Mahony, Mark M; Henneberger, Ruth; Selvin, Joseph; Kennedy, Jonathan; Doohan, Fiona; Marchesi, Julian R; Dobson, Alan D W

    2015-01-01

    A functional metagenomics based approach exploiting the microbiota of suppressive soils from an organic field site has succeeded in the identification of a clone with the ability to inhibit the growth of Bacillus subtilis DSM10. Sequencing of the fosmid identified a putative β-lactamase-like gene abgT. Transposon mutagenesis of the abgT gene resulted in a loss in ability to inhibit the growth of B. subtilis DSM10. Further analysis of the deduced amino acid sequence of AbgT revealed moderate homology to esterases, suggesting that the protein may possess hydrolytic activity. Weak lipolytic activity was detected; however the clone did not appear to produce any β-lactamase activity. Phylogenetic analysis revealed the protein is a member of the family VIII group of lipase/esterases and clusters with a number of proteins of metagenomic origin. The abgT gene was sub-cloned into a protein expression vector and when introduced into the abgT transposon mutant clones restored the ability of the clones to inhibit the growth of B. subtilis DSM10, clearly indicating that the abgT gene is involved in the antibacterial activity. While the precise role of this protein has yet to fully elucidated, it may be involved in the generation of free fatty acid with antibacterial properties. Thus functional metagenomic approaches continue to provide a significant resource for the discovery of novel functional proteins and it is clear that hydrolytic enzymes, such as AbgT, may be a potential source for the development of future antimicrobial therapies. PMID:25692994

  12. Inhibition of the growth of Bacillus subtilis DSM10 by a newly discovered antibacterial protein from the soil metagenome.

    PubMed

    O'Mahony, Mark M; Henneberger, Ruth; Selvin, Joseph; Kennedy, Jonathan; Doohan, Fiona; Marchesi, Julian R; Dobson, Alan D W

    2015-01-01

    A functional metagenomics based approach exploiting the microbiota of suppressive soils from an organic field site has succeeded in the identification of a clone with the ability to inhibit the growth of Bacillus subtilis DSM10. Sequencing of the fosmid identified a putative β-lactamase-like gene abgT. Transposon mutagenesis of the abgT gene resulted in a loss in ability to inhibit the growth of B. subtilis DSM10. Further analysis of the deduced amino acid sequence of AbgT revealed moderate homology to esterases, suggesting that the protein may possess hydrolytic activity. Weak lipolytic activity was detected; however the clone did not appear to produce any β-lactamase activity. Phylogenetic analysis revealed the protein is a member of the family VIII group of lipase/esterases and clusters with a number of proteins of metagenomic origin. The abgT gene was sub-cloned into a protein expression vector and when introduced into the abgT transposon mutant clones restored the ability of the clones to inhibit the growth of B. subtilis DSM10, clearly indicating that the abgT gene is involved in the antibacterial activity. While the precise role of this protein has yet to fully elucidated, it may be involved in the generation of free fatty acid with antibacterial properties. Thus functional metagenomic approaches continue to provide a significant resource for the discovery of novel functional proteins and it is clear that hydrolytic enzymes, such as AbgT, may be a potential source for the development of future antimicrobial therapies.

  13. Effects of metal oxide nanoparticles on the structure and activity of lysozyme.

    PubMed

    Cheng, Yu-Hong; Lai, Chia-Min; Lin, Kuen-Song; Wang, Steven S-S

    2017-03-01

    We investigated the effects of nanoparticles (NPs) on the structure and activity of hen egg-white lysozyme (HEWL) using CeO2 and ZnO NPs. Our results showed that CeO2 NPs triggered the transition of lysozyme secondary structure from α-helix to β-sheet. CeO2 NPs also induced the hydrophobic region of lysozyme to become exposed to the solvent. In contrast, the secondary structure content and hydrophobic region of lysozyme were only slightly changed in the case of ZnO NPs. In addition, the activity of the lysozyme was observed to decrease upon adsorption on CeO2 NPs, whereas the effect of ZnO NPs on activity was negligible. The glutaraldehyde crosslinking results indicated that the percentage of the dimeric form of lysozyme was greatly enhanced by the addition of both NPs. Furthermore, the adsorption capacity, degree of favorability of adsorption, and surface heterogeneity for CeO2 NPs were found to be greater than those on ZnO NPs. Given that CeO2 NPs exhibit a higher surface area/mass than ZnO NPs, the surface concentration of lysozyme on CeO2 NPs was lower than that on ZnO NPs. This result suggested that more direct interactions were involved between CeO2 NPs and lysozyme, thereby leading to a more significant effect. Moreover, higher surface curvatures may also cause destruction of lysozyme's structure and thus affect its activity. In addition, taking into account the surface properties and protein properties, the Toth adsorption model along with the generated site energy distribution was further used to exaplain the difference between the results (e.g., structure, stability, and activity) of lysozyme adsorption on CeO2 and ZnO NPs. The results reported here may aid in better understanding the beneficial or harmful impacts of nanoparticles on the biological systems.

  14. A direct calorimetric determination of denaturation enthalpy for lysozyme in sodium dodecyl sulfate.

    PubMed

    Behbehani, G Rezaei; Saboury, A A; Taleshi, E

    2008-02-15

    Thermodynamics of the interaction between sodium dodecyl sulfate (SDS) with lysozyme were investigated at pH 7.0 and 27 degrees C in phosphate buffer by isothermal titration calorimetry. A new method to follow protein denaturation, and the effect of surfactants on the stability of proteins was introduced. The new solvation model was used to reproduce the enthalpies of lysozyme-SDS interaction over the whole range of SDS concentrations. The solvation parameters recovered from the new equation, attributed to the structural change of lysozyme and its biological activity. At low concentrations of SDS, the binding is mainly electrostatic, with some simultaneous interaction of the hydrophobic tail with nearby hydrophobic patches on the lysozyme. These initial interactions presumably cause some protein unfolding and expose additional hydrophobic sites. The enthalpy of denaturation is 160.81+/-0.02 kJ mol(-1) for SDS.

  15. Does Warming a Lysozyme Solution Cook Ones Data?

    NASA Technical Reports Server (NTRS)

    Pusey, Marc; Burke, Michael; Judge, Russell

    2000-01-01

    Chicken egg white lysozyme has a well characterized thermally driven phase transition. Between pH 4.0 and 5.2, the transition temperature, as defined by the point where the tetragonal and orthorhombic solubility are equal, is a function of the pH, salt (precipitant) type and concentration, and most likely of the buffer concentration as well. This phase transition can be carried out with protein solution alone, prior to the initiation of the crystallization process. We have now measured the kinetics of this process and investigated its reversibility. An aliquot of a stock protein solution is held at a given temperature, and at periodic intervals used to set up batch crystallization experiments. The batch solutions were incubated at 20 C until macroscopic crystals were obtained, at which point the number of crystals in each well were counted. The transition effects increased with temperature, slowly falling off at 30 C with a half time (time to approx. 1/2 the t = 0 number of crystals) of approx. 5 hours, and an estimated half time of approx. 0.5 hours at 43 C. Further, the process was not reversible by simple cooling. After holding a lysozyme solution at 37 C (prior to addition of precipitant) for 16 hours, then cooling and holding it at 4 C, no return to the pre-warmed nucleation kinetics are observed after at least 4 weeks. Thus every thermal excursion above the phase transition point results in a further decrease in the nucleation rate of that solution, the extent being a function of the time and temperature. Orthorhombic lysozyme crystals apparently do not undergo the flow-induced growth cessation of tetragonal lysozyme crystals. We have previously shown that putting the protein in the orthorhombic form does not affect the averaged face growth kinetics, only nucleation, for tetragonal crystals. We may be able to use this differential behavior to elucidate how flow affects tile lysozyme crystal growth process.

  16. Comparative evaluation of multi-purpose solutions in the stabilization of tear lysozyme.

    PubMed

    Barniak, Vicki L; Burke, Susan E; Venkatesh, Srini

    2010-12-01

    The range and extent of tear proteins removed by various multi-purpose solutions has been investigated, but there is little information in the literature about their ability to prevent denaturation of tear proteins, particularly lysozyme. The purpose of this study was to determine the ability of Bausch+Lomb Biotrue™ multi-purpose solution and other care solutions to affect denaturation of lysozyme using a lysozyme activity assay. The test solutions used were: Biotrue multi-purpose solution, Bausch+Lomb renu(®) fresh™, formerly ReNu MultiPlus(®), Alcon OPTI-FREE RepleniSH, Alcon OPTI-FREE EXPRESS, CIBA VISION AQuify, and AMO COMPLETE Multi-Purpose Solution Easy Rub Formula. A phosphate-buffered saline (PBS) solution served as a control. The test and control solutions containing lysozyme were exposed to sodium dodecyl sulfate (SDS), a known denaturant of the enzyme. The assay was based on digestion of the cell wall of Micrococcus luteus in a suspension, a substrate sensitive to active lysozyme. Enzymatic activity against M. luteus was used to assess activity of lysozyme. The decrease in the turbidity of the cell wall suspension, a measure of relative enzyme activity, was determined by following the decrease in absorbance (at 450nm) over time using a spectrophotometer. Statistically significant greater stabilization of lysozyme was observed with Biotrue multi-purpose solution and renu fresh than with OPTI-FREE RepleniSH, OPTI-FREE EXPRESS, AQuify, COMPLETE Multi-Purpose Solution Easy Rub Formula, and a PBS control. The lysozyme activity assay revealed that Biotrue multi-purpose solution and renu fresh have the ability to stabilize lysozyme under conditions that typically denature the protein.

  17. Improvement on the crystallization of lysozyme in the presence of hydrophilic ionic liquid.

    PubMed

    Chen, Xuwei; Ji, Yanpei; Wang, Jianhua

    2010-09-01

    The crystallization of lysozyme with hydrophilic ionic liquid 1,3-butylimidazolium chloride (BBimCl) as an additive was investigated with hanging-drop vapor diffusion crystallization protocol. The elevated threshold to super-saturation caused by the increased solubility of lysozyme in the presence of BBimCl and the slower super-saturation process of lysozyme induced by the negligible vapor pressure of BBimCl contributed to a lower super-saturation degree, offering a promoted ambient circumstance for nucleation and providing a controlled velocity for the growth of lysozyme crystal. These eventually offer a prominent promotion to the crystallization of lysozyme, i.e., less crystal polymorphism and precipitation while larger crystals and significantly improved the tolerance to the concomitant impurities or sample matrices for the crystallization of lysozyme. Therefore, the presence of BBimCl enables the direct crystallization of lysozyme from a real complex sample matrix, i.e., egg-white, which opens a promising avenue for the development of protein crystallization methodology with ionic liquids as an additive and offers vast potentials for the practical separation/purification of proteins of interest from complex real sample matrices.

  18. Lysozyme-lysozyme and lysozyme-salt interactions in the aqueous saline solution: a new square-well potential.

    PubMed

    Chang, Bong Ho; Bae, Young Chan

    2003-01-01

    We investigate lysozyme-lysozyme and lysozyme-salt interactions in electrolyte solutions using a molecular-thermodynamic model. An equation of state based on the statistical mechanical perturbation theory is applied to describe the interactions. The perturbation term includes a new square-well potential of mean force, which implies the information about the lysozyme surface and salt type. The attractive energy of the potential of mean force is correlated with experimental cloud-point temperatures of lysozyme in various solution conditions. The same attractive energy is used to predict osmotic pressure of a given system with no additional parameters. The new potential shows a satisfactory improvement in understanding the interactions between lysozymes in aqueous salt solutions.

  19. Interactions between single-walled carbon nanotubes and lysozyme.

    PubMed

    Bomboi, F; Bonincontro, A; La Mesa, C; Tardani, F

    2011-03-15

    Dispersions of single-walled and non-associated carbon nanotubes in aqueous lysozyme solution were investigated by analyzing the stabilizing effect of both protein concentration and pH. It was inferred that the medium pH, which significantly modifies the protein net charge and (presumably) conformation, modulates the mutual interactions with carbon nanotubes. At fixed pH, in addition, the formation of protein/nanotube complexes scales with increasing lysozyme concentration. Electrophoretic mobility, dielectric relaxation and circular dichroism were used to determine the above features. According to circular dichroism, lysozyme adsorbed onto nanotubes could essentially retain its native conformation, but the significant amount of free protein does not allow drawing definitive conclusions on this regard. The state of charge and charge distribution around nanotubes was inferred by combining electrophoretic mobility and dielectric relaxation methods. The former gives information on changes in the surface charge density of the complexes, the latter on modifications in the electrical double layer thickness around them. Such results are complementary each other and univocally indicate that some LYS molecules take part to binding. Above a critical protein/nanotube mass ratio, depletion phenomena were observed. They counteract the stabilization mechanism, with subsequent nanotube/nanotube aggregation and phase separation. Protein-based depletion phenomena are similar to formerly reported effects, observed in aqueous surfactant systems containing carbon nanotubes.

  20. Relationship Between Equilibrium Forms of Lysozyme Crystals and Precipitant Anions

    NASA Technical Reports Server (NTRS)

    Nadarajah, Arunan

    1996-01-01

    Molecular forces, such as electrostatic, hydrophobic, van der Waals and steric forces, are known to be important in determining protein interactions. These forces are affected by the solution conditions and changing the pH, temperature or the ionic strength of the solution can sharply affect protein interactions. Several investigations of protein crystallization have shown that this process is also strongly dependent on solution conditions. As the ionic strength of the solution is increased, the initially soluble protein may either crystallize or form an amorphous precipitate at high ionic strengths. Studies done on the model protein hen egg white lysozyme have shown that different crystal forms can be easily and reproducibly obtained, depending primarily on the anion used to desolubilize the protein. In this study we employ pyranine to probe the effect of various anions on the water structure. Additionally, lysozyme crystallization was carried out at these conditions and the crystal form was determined by X-ray crystallography. The goal of the study was to understand the physico-chemical basis for the effect of changing the anion concentration on the equilibrium form of lysozyme crystals. It will also verify the hypothesis that the anions, by altering the bulk water structure in the crystallizing solutions, alter the surface energy of the between the crystal faces and the solution and, consequently, the equilibrium form of the crystals.

  1. Expression and purification of active recombinant equine lysozyme in Escherichia coli.

    PubMed

    Casaite, Vida; Bruzyte, Simona; Bukauskas, Virginijus; Setkus, Arunas; Morozova-Roche, Ludmilla A; Meskys, Rolandas

    2009-11-01

    Equine lysozyme (EL) is a calcium (Ca)-binding lysozyme and is an intermediary link between non-Ca-binding C-type lysozyme and alpha-lactalbumin. The feature of lysozymes to assemble into the fibrils has recently gained considerable attention for the investigation of the functional properties of these proteins. To study the structural and functional properties of EL, a synthetic gene was cloned and EL was overexpressed in Escherichia coli as a fused protein. The His-tagged recombinant EL was accumulated as inclusion bodies. Up to 50 mg/l of the recombinant EL could be achieved after purification by Ni(2+) affinity chromatography, refolding in the presence of arginine, CM-Sepharose column purification following TEV protease cleavage. The purified protein was functionally active, as determined by the lysozyme activity, proving the proper folding of protein. The purified lysozyme was used for the oligomerisation studies. The protein formed amyloid fibrils during incubation in acidic pH and elevated temperature. The recombinant EL forms two types of fibrils: ring shaped and linear, similar to the native EL.

  2. Leucine-rich repeats containing protein functions in the antibacterial immune reaction in stomach of kuruma shrimp Marsupenaeus japonicus.

    PubMed

    Shi, Xiu-Zhen; Feng, Xiao-Wu; Sun, Jie-Jie; Zhao, Xiao-Fan; Wang, Jin-Xing

    2017-02-01

    Leucine rich repeat (LRR) motif exists in many immune receptors of animals and plants. Most LRR containing (LRRC) proteins are involved in protein-ligand and protein-protein interaction, but the exact functions of most LRRC proteins were not well-studied. In this study, an LRRC protein was identified from kuruma shrimp Marsupenaeus japonicus, and named as MjLRRC1. MjLRRC1 was consistently expressed in different tissues of normal shrimp with higher expression in gills and stomach. At the transcriptional level, there were no significant changes of MjLRRC1 after injection of Vibrio anguillarum or Staphylococcus aureus in gills and hepatopancreas. While in V. anguillarum oral infection, MjLRRC1 was upregulated in stomach but not in intestine. The recombinant MjLRRC1 protein could bind to Gram-positive and Gram-negative bacteria, bacterial cell wall components including peptidoglycan, lipoteichoic acid, and lipopolysaccharide. MjLRRC1 regulated the expression of some antimicrobial peptide (AMP) genes and participated in bacteria clearance of stomach. All these results suggested that MjLRRC1 might play important roles in antibacterial immune response of kuruma shrimp.

  3. Lysozyme dimer association: Similarities and differences compared with lysozyme monomer association

    NASA Astrophysics Data System (ADS)

    Onuma, Kazuo; Inaka, Koji

    2008-03-01

    The protein with a molecular weight of 28.6 kDa in lysozyme solution, which has been recognized as a lysozyme dimer, was purified and its association was observed using time-resolved static light scattering and dynamic light scattering under the same buffer condition as that used in lysozyme monomer association. The chromatography results and SDS-PAGE analysis showed that the bonding state of each molecule in a dimer unit was not uniform, i.e., there were at least two kinds of bonds, strong and weak. Some of the weak-bonded dimmers dissociated to monomers (molecular weight: 14.3 kDa) in the SDS-PAGE process. The relative amount of weak-bonded dimers greatly affected the association kinetics. With a 99% pure dimer solution (1% monomers in SDS-PAGE), association proceeded in the same manner as that of a monomer solution: the Zimm-square plot had a concave shape with a maximum at a particular q2 for apparent protein concentrations, up to 2.4 mg/mL. The dynamic light-scattering data showed clear bimodal (dimer and aggregate), distributions. With a 95% pure dimer solution, the association behavior drastically changed when the apparent concentration exceeded 2.0 mg/mL. The Zimm-square plot had a bending point at a low q2, and two discrete lines fitted the plot. The particles in the solution were either oligomers or large aggregates, both of which had polydispersity distributions, and an amorphous phase formed from the aggregates. This was not observed for monomer association.

  4. Activity of lysozyme on Lactobacillus hilgardii strains isolated from Port wine.

    PubMed

    Dias, Rita; Vilas-Boas, Eduardo; Campos, Francisco M; Hogg, Tim; Couto, José António

    2015-08-01

    This work evaluated the effect of lysozyme on lactobacilli isolated from Port wine. Bacterial growth experiments were conducted in MRS/TJ medium and inactivation studies were performed in phosphate buffer (KH2PO4), distilled water and wine supplemented with different concentrations of lysozyme. The response of bacteria to lysozyme was found to be highly strain dependent. Some strains of Lactobacillus hilgardii together with Lactobacillus collinoides and Lactobacillus fructivorans were found to be resistant to concentrations of lysozyme as high as 2000 mg/L. It was observed that among the L. hilgardii taxon the resistant strains possess an S-layer coat. Apparently, the strains of L. collinoides and L. fructivorans studied are also S-layer producers as suggested by the total protein profile obtained by SDS-PAGE. Thus, the hypothetical protective role of the S-layer against the action of lysozyme was investigated. From the various treatments used to remove the protein from the surface of the cells, the one employing LiCl (5 M) was the most effective. LiCl pre-treated cells exposed to lysozyme (2000 mg/L) in KH2PO4 buffer maintained its resistance. However, when cells were suspended in distilled water an increased sensitivity to lysozyme was observed. Moreover, it was found that the addition of ethanol (20% v/v) to the suspension medium (distilled water) triggered a strong inactivation effect especially on cells previously treated with LiCl (reduction of >6 CFU log cycles). The results suggest that the S-layer exerts a protective effect against lysozyme and that the cell suspension medium influences the bacteriolysis efficiency. It was also noted that ethanol enhances the inactivation effect of lysozyme.

  5. Locations of Bromide Ions in Tetragonal Lysozyme Crystals

    NASA Technical Reports Server (NTRS)

    Lim, Kap; Nadarajah, Arunan; Forsythe, Elizabeth L.; Pusey, Marc L.

    1998-01-01

    Anions have been shown to play a dominant role in the crystallization of chicken egg-white lysozyme from salt solutions. Previous studies employing X-ray crystallography have found one chloride ion binding site in the tetragonal crystal form of the protein and four nitrate ion binding sites in the monoclinic form. In this study the anion positions in the tetragonal form were determined from the difference Fourier map obtained from lysozyme crystals grown in bromide and chloride solutions. Five possible anion-binding sites were found in this manner. Some of these sites were in pockets containing basic residues while others were near neutral, but polar, residues. The sole chloride ion binding site found in previous studies was confirmed, while four further sites were found which corresponded to the four binding sites found for nitrate ions in monoclinic crystals. The study suggests that most of the anion-binding sites in lysozyme remain unchanged even when different anions and different crystal forms of lysozyme are employed.

  6. Kinetic Roughening Transition and Energetics of Tetragonal Lysozyme Crystal Growth

    NASA Technical Reports Server (NTRS)

    Gorti, Sridhar; Forsythe, Elizabeth L.; Pusey, Marc L.

    2004-01-01

    Interpretation of lysozyme crystal growth rates using well-established physical theories enabled the discovery of a phenomenon possibly indicative of kinetic roughening. For example, lysozyme crystals grown above a critical supersaturation sigma, (where supersaturation sigma = ln c/c(sub eq), c = the protein concentration and c(sub eq) = the solubility concentration) exhibit microscopically rough surfaces due to the continuous addition of growth units anywhere on the surface of a crystal. The rate of crystal growth, V(sub c), for the continuous growth process is determined by the continuous flux of macromolecules onto a unit area of the crystal surface, a, from a distance, xi, per unit time due to diffusion, and a probability of attachment onto the crystal surface, expressed. Based upon models applied, the energetics of lysozyme crystal growth was determined. The magnitudes of the energy barriers of crystal growth for both the (110) and (101) faces of tetragonal lysozyme crystals are compared. Finally, evidence supportive of the kinetic roughening hypothesis is presented.

  7. Locations of Halide Ions in Tetragonal Lysozyme Crystals

    NASA Technical Reports Server (NTRS)

    Lim, Kap; Adimurthy, Ganapathi; Nadarajah, Arunan; Forsythe, Elizabeth L.; Pusey, Marc L.

    1998-01-01

    Anions play an important role in the crystallization of lysozyme, and are known to bind to the crystalline protein. Previous studies employing X-ray crystallography had found one chloride ion binding site in the tetragonal crystal form of the protein and four nitrate ion binding sites in the monoclinic form. Studies using other approaches have reported more chloride ion binding sites, but their locations were not known. Knowing the precise location of these anions is also useful in determining the correct electrostatic fields surrounding the protein. In the first part of this study the anion positions in the tetragonal form were determined from the difference Fourier map obtained from the lysozyme crystals grown in bromide and chloride solutions under identical conditions. The anion locations were then obtained from standard crystallographic methods and five possible anion binding sites were found in this manner. The sole chloride ion binding site found in previous studies was confirmed. The remaining four sites were new ones for tetragonal lysozyme crystals. However, three of these new sites and the previously found one corresponded to the four unique binding sites found for nitrate ions in monoclinic crystals. This suggests that most of the anion binding sites in lysozyme remain unchanged, even when different anions and different crystal forms of lysozyme are employed. It is unlikely that there are many more anions in the tetragonal lysozyme crystal structure. Assuming osmotic equilibrium it can be shown that there are at most three more anions in the crystal channels. Some of the new anion binding sites found in this study were, as expected, in pockets containing basic residues. However, some of them were near neutral, but polar, residues. Thus, the study also showed the importance of uncharged, but polar groups, on the protein surface in determining its electrostatic field. This was important for the second part of this study where the electrostatic field

  8. Unfolding mechanism of lysozyme in various urea solutions: Insights from fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Chen, Bang; Zhang, Hongjia; Xi, Wenying; Zhao, Liqing; Liang, Li; Chen, Yantao

    2014-11-01

    Fluorescence spectroscopic technique is very popular in exploring the folding/unfolding process of proteins. In this paper, unfolding process of hen egg-white lysozyme was investigated in various denaturing solutions. Firstly, polymer solution theory was employed to comprehend the dependence of fluorescence quenching effect on protein concentration, and dynamic contact concentration was suggested as a critical value for related fluorescence experiment. Secondly, it was found that urea alone could not completely unfold lysozyme but did when together with DTT or HCl. Lysozyme was destabilized in concentrated urea solution, but still could maintain its spatial structure. Phase diagram of fluorescence intensities revealed that HCl could enhance the denaturing capacity of urea, resulting in the emergence of intermediate state in the thermodynamic unfolding process of lysozyme.

  9. In vitro antioxidant and antibacterial properties of hydrolysed proteins of delimed tannery fleshings: comparison of acid hydrolysis and fermentation methods.

    PubMed

    Balakrishnan, Bijinu; Prasad, Binod; Rai, Amit Kumar; Velappan, Suresh Puthanveetil; Subbanna, Mahendrakar Namadev; Narayan, Bhaskar

    2011-04-01

    Proteins in delimed tannery fleshings were fermentatively hydrolysed using Enterococcus faecium NCIM5335 and also hydrolysed using mild organic acids (formic acid and propionic acid). The liquor portion containing hydrolysed proteins was spray dried, in both the cases, to obtain a powder. The spray dried powder was evaluated for in vitro antioxidant activities with respect to scavenging different free radicals and antibacterial properties against nine different pathogens. Fermentation and acid hydrolysates scavenged 83 and 75.3% of 2,2-azino-bis-3-ethyl-benzthiazoline-6-sulphonic acid (ABTS) radicals, respectively, at a protein concentration of 0.25 mg. Further, fermentation hydrolysate showed higher 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity of 59% as compared to 56% scavenging by acid hydrolysate at a protein concentration of 5 mg. Acid hydrolysate exhibited lesser (82.3%) peroxy radical scavenging compared to hydrolysate from fermentation (88.2%) at a protein concentration of 10 mg. However, acid hydrolysate exhibited higher (89.2%) superoxide anion scavenging while its fermentation counterpart showed lower activity (85.4%) at 2.5 mg hydrolysate protein. Well as superoxide anion scavenging properties. All the in vitro antioxidant properties exhibited dose dependency. Fermentation hydrolysate exhibited maximum antagonistic activity against Salmonella typhi FB231, from among host of pathogens evaluated. Both the hydrolysates have potential to be ingredients in animal feeds and can help reduce oxidative stress in the animals.

  10. Investigating the effects of erythrosine B on amyloid fibril formation derived from lysozyme.

    PubMed

    Kuo, Chun-Tien; Chen, Yi-Lin; Hsu, Wei-Tse; How, Su-Chun; Cheng, Yu-Hong; Hsueh, Shu-Shun; Liu, Hwai-Shen; Lin, Ta-Hsien; Wu, Josephine W; Wang, Steven S-S

    2017-05-01

    Formation of amyloid fibrils has been associated with at least 30 different protein aggregation diseases. The 129-residue polypeptide hen lysozyme, which is structurally homologous to human lysozyme, has been demonstrated to exhibit amyloid fibril-forming propensity in vitro. This study is aimed at exploring the influence of erythrosine B on the in vitro amyloid fibril formation of hen lysozyme at pH 2.0 and 55°C using ThT binding assay, transmission electron microscopy, far-UV circular dichroism absorption spectroscopy, 1-anilinonaphthalene-8-sulfonic acid fluorescence spectroscopy, and synchronous fluorescence study. We found that lysozyme fibrillogenesis was dose-dependently suppressed by erythrosine B. In addition, our far-UV CD and ANS fluorescence data showed that, as compared with the untreated lysozyme control, the α-to-ß transition and exposure of hydrophobic clusters in lysozyme were reduced upon treatment with erythrosine B. Moreover, it could be inferred that the binding of erythrosine B occurred in the vicinity of the tryptophan residues. Finally, molecular docking and molecular dynamics simulations were further employed to gain some insights into the possible binding site(s) and interactions between lysozyme and erythrosine B. We believe the results obtained here may contribute to the development of potential strategies/approaches for the suppression of amyloid fibrillogenesis, which is implicated in amyloid pathology.

  11. Influence of graphene oxide and reduced graphene oxide on the activity and conformation of lysozyme.

    PubMed

    Bai, Yitong; Ming, Zhu; Cao, Yuye; Feng, Shicheng; Yang, Hua; Chen, Lingyun; Yang, Sheng-Tao

    2017-03-08

    The dramatically different bio-effects of graphene and graphene oxide (GO) have been widely observed in diverse biological systems, which determine the applications and toxicity of graphene materials. To elucidate the mechanism at molecular level, it is urgent to investigate the enzyme-graphene interaction and its consequences. In this study, we comparatively studied the influence of GO and reduced GO (RGO) on the activity and conformation of lysozyme to provide better understandings of their different bio-effects. Both GO and RGO adsorbed large quantities of lysozyme after incubation. GO inhibited lysozyme activity seriously, while RGO nearly had no influence on the enzyme activity. The different inhibitions of enzyme activity could be explained by the lysozyme conformational changes, where GO induced more changes to the protein conformation according to UV-vis absorbance, far-UV circular dichroism spectra, intrinsic fluorescence quenching, and infrared spectra. Based on the spectroscopic changes of lysozyme, GO induced the loss of secondary structure and exposed the active site of lysozyme more to the aqueous environment. In addition, neither GO nor RGO induced the fibrillation of lysozyme after 12d incubation. The results collectively indicated that the oxidation degree significantly impacted the enzyme-graphene interaction. The implications to the designs of enzyme-graphene system for bio-related applications and the toxicological effects of graphene materials are discussed.

  12. Control of Electrostatic Interactions Between F-Actin And Genetically Modified Lysozyme in Aqueous Media

    SciTech Connect

    Sanders, L.K.; Xian, W.; Guaqueta, C.; Strohman, M.; Vrasich, C.R.; Luijten, E.; Wong, G.C.L.

    2009-06-04

    The aim for deterministic control of the interactions between macroions in aqueous media has motivated widespread experimental and theoretical work. Although it has been well established that like-charged macromolecules can aggregate under the influence of oppositely charged condensing agents, the specific conditions for the stability of such aggregates can only be determined empirically. We examine these conditions, which involve an interplay of electrostatic and osmotic effects, by using a well defined model system composed of F-actin, an anionic rod-like polyelectrolyte, and lysozyme, a cationic globular protein with a charge that can be genetically modified. The structure and stability of actin-lysozyme complexes for different lysozyme charge mutants and salt concentrations are examined by using synchrotron x-ray scattering and molecular dynamics simulations. We provide evidence that supports a structural transition from columnar arrangements of F-actin held together by arrays of lysozyme at the threefold interstitial sites of the actin sublattice to marginally stable complexes in which lysozyme resides at twofold bridging sites between actin. The reduced stability arises from strongly reduced partitioning of salt between the complex and the surrounding solution. Changes in the stability of actin-lysozyme complexes are of biomedical interest because their formation has been reported to contribute to the persistence of airway infections in cystic fibrosis by sequestering antimicrobials such as lysozyme. We present x-ray microscopy results that argue for the existence of actin-lysozyme complexes in cystic fibrosis sputum and demonstrate that, for a wide range of salt conditions, charge-reduced lysozyme is not sequestered in ordered complexes while retaining its bacterial killing activity.

  13. Control of electrostatic interactions between F-actin and genetically modified lysozyme in aqueous media

    SciTech Connect

    Sanders, Lori K.; Xian, Wujing; Guaqueta, Camilo; Strohman, Michael J.; Vrasich, Chuck R.; Luijten, Erik; Wong, Gerard C.L.

    2008-07-11

    The aim for deterministic control of the interactions between macroions in aqueous media has motivated widespread experimental and theoretical work. Although it has been well established that like-charged macromolecules can aggregate under the influence of oppositely charged condensing agents, the specific conditions for the stability of such aggregates can only be determined empirically. We examine these conditions, which involve an interplay of electrostatic and osmotic effects, by using a well defined model system composed of F-actin, an anionic rod-like polyelectrolyte, and lysozyme, a cationic globular protein with a charge that can be genetically modified. The structure and stability of actin-lysozyme complexes for different lysozyme charge mutants and salt concentrations are examined by using synchrotron x-ray scattering and molecular dynamics simulations. We provide evidence that supports a structural transition from columnar arrangements of F-actin held together by arrays of lysozyme at the threefold interstitial sites of the actin sublattice to marginally stable complexes in which lysozyme resides at twofold bridging sites between actin. The reduced stability arises from strongly reduced partitioning of salt between the complex and the surrounding solution. Changes in the stability of actin-lysozyme complexes are of biomedical interest because their formation has been reported to contribute to the persistence of airway infections in cystic fibrosis by sequestering antimicrobials such as lysozyme. We present x-ray microscopy results that argue for the existence of actin-lysozyme complexes in cystic fibrosis sputum and demonstrate that, for a wide range of salt conditions, charge-reduced lysozyme is not sequestered in ordered complexes while retaining its bacterial killing activity.

  14. Lysozyme Catalyzes the Formation of Antimicrobial Silver Nanoparticles

    DTIC Science & Technology

    2009-04-03

    Guo, C.; Sun, Y. Synthesis , Characterization, and Self - Assembly of Protein Lysozyme Monolayer-Stabilized Gold Nanoparticles. Langmuir 2007, 23, 10533...to achieve with aqueous-based synthesis , because water facilitates strong ionic interactions between forming particles.25 The hydrophobicity of...2002, 1, 169–172. 16. Xie, J.; Lee, J. Y.; Wang, D. I. C.; Ting, Y. P. Silver Nanoplates : From Biological to Biomimetic Synthesis . ACS Nano 2007, 1

  15. Lysozyme Catalyzes the Formation of Antimicrobial Silver Nanoparticles (POSTPRINT)

    DTIC Science & Technology

    2009-04-01

    Y. Synthesis , Characterization, and Self - Assembly of Protein Lysozyme Monolayer-Stabilized Gold Nanoparticles. Langmuir 2007, 23, 10533–10538. 28...aqueous-based synthesis , because water facilitates strong ionic interactions between forming particles.25 The hydrophobicity of methanol is a major factor...172. 16. Xie, J.; Lee, J. Y.; Wang, D. I. C.; Ting, Y. P. Silver Nanoplates : From Biological to Biomimetic Synthesis . ACS Nano 2007, 1, 429–439. 17

  16. Water in hydrated orthorhombic lysozyme crystal: Insight from atomistic simulations.

    PubMed

    Hu, Zhongqiao; Jiang, Jianwen; Sandler, Stanley I

    2008-08-21

    Biologically important water in orthorhombic lysozyme crystal is investigated using atomistic simulations. A distinct hydration shell surrounding lysozyme molecules is found from the number distribution of water molecules. While the number of water molecules in the hydration shell increases, the percentage decreases as the hydration level rises. Adsorption of water in the lysozyme crystal shows type-IV behavior. At low hydration levels, water molecules primarily intercalate the minor pores and cavity in the crystal due to the strong affinity between protein and water. At high hydration levels, the major pores are filled with liquidlike water as capillary condensation occurs. A type-H4 hysteresis loop is observed in the adsorption and desorption isotherms. The locations of the water molecules identified from simulation match fairly well with the experimentally determined crystallographic hydration sites. As observed in experiment, water exhibits anomalous subdiffusion because of the geometric restrictions and interactions of protein. With increasing hydration level, this anomaly is reduced and the diffusion of water tends to progressively approach normal Brownian diffusion. The flexibility of protein framework slightly enhances water mobility, but this enhancement decreases with increasing hydration level.

  17. Water in hydrated orthorhombic lysozyme crystal: Insight from atomistic simulations

    NASA Astrophysics Data System (ADS)

    Hu, Zhongqiao; Jiang, Jianwen; Sandler, Stanley I.

    2008-08-01

    Biologically important water in orthorhombic lysozyme crystal is investigated using atomistic simulations. A distinct hydration shell surrounding lysozyme molecules is found from the number distribution of water molecules. While the number of water molecules in the hydration shell increases, the percentage decreases as the hydration level rises. Adsorption of water in the lysozyme crystal shows type-IV behavior. At low hydration levels, water molecules primarily intercalate the minor pores and cavity in the crystal due to the strong affinity between protein and water. At high hydration levels, the major pores are filled with liquidlike water as capillary condensation occurs. A type-H4 hysteresis loop is observed in the adsorption and desorption isotherms. The locations of the water molecules identified from simulation match fairly well with the experimentally determined crystallographic hydration sites. As observed in experiment, water exhibits anomalous subdiffusion because of the geometric restrictions and interactions of protein. With increasing hydration level, this anomaly is reduced and the diffusion of water tends to progressively approach normal Brownian diffusion. The flexibility of protein framework slightly enhances water mobility, but this enhancement decreases with increasing hydration level.

  18. Adsorption and desorption studies of lysozyme by Fe3O4-polymer nanocomposite via fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Koc, Kenan; Alveroglu, Esra

    2015-06-01

    The work have been undertaken in this study is to synthesis and characterize Fe3O4-polymer nanocomposites which are having different morphological properties. Also, investigation of the adsorption and desorption behaviour of lysozyme onto Fe3O4-polymer nanocomposites have been studied. Fe3O4 nanoparticles, synthesized by in situ in polyacrylamide hydrogels, show super-paramagnetic behaviour and saturation magnetization of composite material have been tuned by changing the hydrogel conformation. Adsorption and desorption studies of lysozyme were followed by using pure water at room temperature via fluorescence measurements. Fluorescence measurements showed that, the composite materials adsorbed lysozyme molecules less than 20 s and higher monomer concentration of composite materials cause faster adsorption. Besides, structure of lysozyme molecules were not changed during the adsorption and desorption. As a result Fe3O4-polymer nanocomposites could be used for drug delivery, protein separation and PAAm gels could be used for synthesis of magnetic composites with varying magnetic properties.

  19. Dose‑dependent effect of lysozyme upon Candida albicans biofilm.

    PubMed

    Sebaa, Sarra; Hizette, Nicolas; Boucherit-Otmani, Zahia; Courtois, Philippe

    2017-03-01

    The present study investigated the in vitro effect of lysozyme (0-1,000 µg/ml) on Candida albicans (C. albicans) biofilm development. Investigations were conducted on C. albicans ATCC 10231 and on 10 clinical isolates from dentures. Strains were cultured aerobically at 37˚C in Sabouraud broth. Yeast growth was evaluated by turbidimetry. Biofilm biomass was quantified on a polystyrene support by crystal violet staining and on acrylic surfaces by counts of colony forming units. Lysozyme affected biofilm formation to a greater extent than it affected growth. For the ATCC 10231 reference strain, lysozyme acted as a biofilm promotor on polystyrene at the highest concentration tested (1,000 µg/ml, non‑physiological). When the reference strain was investigated on acrylic resin support, lysozyme acted as a significant biofilm promotor on rough resin, but less on smooth resin. The attached biomass in the presence of physiological concentrations of lysozyme (10‑30 µg/ml) was significantly decreased compared with the hypothetical value of 100% using a one‑sample t‑test, but a comparison between the different lysozyme conditions using analysis of variance and post hoc tests did not reveal significant differences. In 10 wild strains, different patterns of biofilm formation on polystyrene were observed in the presence of lysozyme. Some strains, characterized by large amounts of biofilm formation in the presence of 1,000 µg/ml lysozyme, were poor biofilm producers at low concentrations of lysozyme. In contrast, some strains that were poor biofilm producers with a high lysozyme concentration were more inhibited by low concentrations of lysozyme. The present study emphasizes the need to develop strategies for biofilm control based on in vitro experiments, and to implement these in clinical trials prior to approval of hygiene products enriched with exocrine proteins, such as lysozyme. Further studies will extend these investigations to other Candida species

  20. Relationship of Salivary Lactoferrin and Lysozyme Concentrations with Early Childhood Caries

    PubMed Central

    Moslemi, Masoumeh; Sattari, Mandana; Kooshki, Fahimeh; Fotuhi, Faezeh; Modarresi, Neda; Khalili Sadrabad, Zahra; Shadkar, Mohammad Saeid

    2015-01-01

    Background and aims. Lysozyme and lactoferrin are salivary proteins which play an important role in innate defense mechanisms against bacteria. This study investigated the association of salivary lysozyme and lactoferrin concentrations with early childhood caries (ECC). Materials and methods. This study was carried out on 42 healthy children (age range, 36 to 71 months), of whom 21 were caries free (CF) and 21 had ECC. Disposable needle-less syringes were used to collect unstimulated saliva from buccal and labial vestibules. Fifteen children who had ECC were treated completely and their saliva was collected in the same way for the second time, three months after treatment. Lysozyme and lactoferrin concentrations were measured and recorded by the ELISA method. The intergroup comparisons were carried out using chi-square, Student’s t-test and Wilcoxon signed ranked test. A P-value less than 0.05 was considered as statistically significant. Results. The mean concentration of lysozyme was significantly higher in CF group compared with that of ECC group (P = 0.04). Although the mean concentration of lactoferrin in ECC group was higher in comparison with ECC group, the difference was not statistically significant (P = 0.06). After dental treatment, the mean concentrations of lysozyme and lactoferrin did not change in comparison with their concentrations before treatment. Conclusion. ECC may have a relationship with lower concentrations of unstimulated salivary lactoferrin and lysozyme and reduced amounts of these two salivary proteins may be a risk factor for dental caries in children. PMID:26236438

  1. Interaction of nonionic detergents with the specific sites of lysozyme amyloidogenic region - inhibition of amyloid fibrillization.

    PubMed

    Siposova, Katarina; Kozar, Tibor; Musatov, Andrey

    2017-02-01

    Two nonionic detergents, Triton X-100 (TX-100) and n-dodecyl-β-d-maltoside (DDM) were tested for their ability to affect lysozyme amyloid aggregation. We have demonstrated that fibrillization of lysozyme is completely inhibited by low sub-micellar concentrations of both of these detergents. The apparent IC50 values were calculated to be 22μM and 26μM for TX-100 and DDM, respectively. The detergent/protein ratio is not the only parameter controlling inhibition. The precise timing of the detergent addition was found to be also crucial. It appears that the primary inhibitory activity of detergents resulted from inhibition of nuclei formation, in addition to inhibition of fibril polymerization at the early stage of protofibrils growth. The docking study revealed that Asn-59, Trp-63 and Ala-107, all present within the lysozyme amyloidogenic region, were involved in the interaction with both detergents. In addition, TX-100 also interacted with Gln-57 and Asp-103 within lysozyme. Moreover, based on our computational results, TX-100 bridges the Gln-57 and Ala-107 amino acids of the amyloidogenic segment of lysozyme and therefore inhibits more effectively the amyloid fibril formation. Along these lines, the knowledge gained from our study indicates that the detergents or their derivatives may be applicable as a promising strategy for the modulation of lysozyme protein aggregation.

  2. Characterization of Bioactive Recombinant Human Lysozyme Expressed in Milk of Cloned Transgenic Cattle

    PubMed Central

    Yang, Bin; Wang, Jianwu; Tang, Bo; Liu, Yufang; Guo, Chengdong; Yang, Penghua; Yu, Tian; Li, Rong; Zhao, Jianmin; Zhang, Lei; Dai, Yunping; Li, Ning

    2011-01-01

    Background There is great potential for using transgenic technology to improve the quality of cow milk and to produce biopharmaceuticals within the mammary gland. Lysozyme, a bactericidal protein that protects human infants from microbial infections, is highly expressed in human milk but is found in only trace amounts in cow milk. Methodology/Principal Findings We have produced 17 healthy cloned cattle expressing recombinant human lysozyme using somatic cell nuclear transfer. In this study, we just focus on four transgenic cattle which were natural lactation. The expression level of the recombinant lysozyme was up to 25.96 mg/L, as measured by radioimmunoassay. Purified recombinant human lysozyme showed the same physicochemical properties, such as molecular mass and bacterial lysis, as its natural counterpart. Moreover, both recombinant and natural lysozyme had similar conditions for reactivity as well as for pH and temperature stability during in vitro simulations. The gross composition of transgenic and non-transgenic milk, including levels of lactose, total protein, total fat, and total solids were not found significant differences. Conclusions/Significance Thus, our study not only describes transgenic cattle whose milk offers the similar nutritional benefits as human milk but also reports techniques that could be further refined for production of active human lysozyme on a large scale. PMID:21436886

  3. Amphiphilic copolymers reduce aggregation of unfolded lysozyme more effectively than polyethylene glycol

    NASA Astrophysics Data System (ADS)

    Chin, Jaemin; Mustafi, Devkumar; Poellmann, Michael J.; Lee, Raphael C.

    2017-02-01

    Certain amphiphilic block copolymers are known to prevent aggregation of unfolded proteins. To better understand the mechanism of this effect, the optical properties of heat-denatured and dithiothreitol reduced lysozyme were evaluated with respect to controls using UV–Vis spectroscopy, transmission electron microscopy (TEM) and circular dichroism (CD) measurements. Then, the effects of adding Polyethylene Glycol (8000 Da), the triblock surfactant Poloxamer 188 (P188), and the tetrablock copolymer Tetronic 1107 (T1107) to the lysozyme solution were compared. Overall, T1107 was found to be more effective than P188 in inhibiting aggregation, while PEG exhibited no efficacy. TEM imaging of heat-denatured and reduced lysozymes revealed spherical aggregates with on average 250–450 nm diameter. Using CD, more soluble lysozyme was recovered with T1107 than P188 with β-sheet secondary structure. The greater effectiveness of the larger T1107 in preventing aggregation of unfolded lysozyme than the smaller P188 and PEG points to steric hindrance at play; signifying the importance of size match between the hydrophobic region of denatured protein and that of amphiphilic copolymers. Thus, our results corroborate that certain multi-block copolymers are effective in preventing heat-induced aggregation of reduced lysozymes and future studies warrant more detailed focus on specific applications of these copolymers.

  4. Modification of Martini force field for molecular dynamics simulation of hydrophobic charge induction chromatography of lysozyme.

    PubMed

    Zhang, Lin; Bai, Shu; Sun, Yan

    2011-06-01

    Modeling, especially the force field, is crucial for the accuracy of molecular dynamics (MD) simulations. In order for more accurate description of adsorption and desorption behaviors of lysozyme in hydrophobic charge induction chromatography (HCIC), the Martini coarse-grained (CG) force field has been modified based on the statistical analysis and comparison of an all-atom (AA) force field, GROMOS96 43A1, and the Martini force field. The parameters describing the protein-adsorbent interactions have been adjusted to avoid too strong and unrealistic adsorption of lysozyme on the agarose matrix and HCIC ligands. It is found that the adsorption and desorption behaviors monitored using the modified Martini force field and MD simulation are consistent with previous simulation results with 46-bead β-barrel model protein. Repeated adjustment of both protein position and orientation is necessary to generate enough contacts for a stable adsorption. After reducing the pH in the mobile phase, the lysozyme-ligand electrostatic repulsion leads to protein desorption. In the adsorption process, little conformational transition of lysozyme is observed due to its stable structure, which is in line with previous experimental observations. So, it is concluded that after appropriate modification, the Martini force field can be used to examine the HCIC process of lysozyme. The modification strategy has thus extended the applicability of the Martini force field to protein chromatography, and it is expected to facilitate studies of exploring the molecular details in adsorption chromatography of proteins.

  5. Evaluation of alternatives for human lysozyme purification from transgenic rice: impact of phytic acid and buffer.

    PubMed

    Wilken, Lisa R; Nikolov, Zivko L

    2010-01-01

    Producing economically competitive recombinant human lysozyme from transgenic rice demands an inexpensive purification process for nonpharmaceutical applications. Human lysozyme is a basic protein, and thus, cation exchange chromatography was the selected method for lysozyme purification. Similar to other protein production systems, the identification of critical impurities in the rice extract was important for the development of an efficient purification process. Previous adsorption data indicated that phytic acid was probably responsible for an unacceptably low cation exchange adsorption capacity. In this study, we confirm that reducing phytic acid concentration improves lysozyme binding capacity and investigate alternative process conditions that reduce phytic acid interference. Compared with the previous best process, the adsorption capacity of human lysozyme was increased from 8.6 to 19.7 mg/mL when rice extract was treated with phytase to degrade phytic acid. Using tris buffer to adjust pH 4.5 extract to pH 6 before adsorption reduced phytic acid interference by minimizing phytic acid-lysozyme interactions, eliminated the need for phytase treatment, and increased the binding capacity to 25 mg/mL. Another method of reducing phytic acid concentration was to extract human lysozyme from rice flour at pH 10 with 50 mM NaCl in 50 mM sodium carbonate buffer. A similar binding capacity (25.5 mg/mL) was achieved from pH 10 extract that was clarified by acidic precipitation and adjusted to pH 6 for adsorption. Lysozyme purities ranged from 95 to 98% for all three processing methods. The tris-mediated purification was the most efficient of the alternatives considered.

  6. Oral mucosal lipids are antibacterial against Porphyromonas gingivalis, induce ultrastructural damage, and alter bacterial lipid and protein compositions.

    PubMed

    Fischer, Carol L; Walters, Katherine S; Drake, David R; Dawson, Deborah V; Blanchette, Derek R; Brogden, Kim A; Wertz, Philip W

    2013-09-01

    Oral mucosal and salivary lipids exhibit potent antimicrobial activity for a variety of Gram-positive and Gram-negative bacteria; however, little is known about their spectrum of antimicrobial activity or mechanisms of action against oral bacteria. In this study, we examine the activity of two fatty acids and three sphingoid bases against Porphyromonas gingivalis, an important colonizer of the oral cavity implicated in periodontitis. Minimal inhibitory concentrations, minimal bactericidal concentrations, and kill kinetics revealed variable, but potent, activity of oral mucosal and salivary lipids against P. gingivalis, indicating that lipid structure may be an important determinant in lipid mechanisms of activity against bacteria, although specific components of bacterial membranes are also likely important. Electron micrographs showed ultrastructural damage induced by sapienic acid and phytosphingosine and confirmed disruption of the bacterial plasma membrane. This information, coupled with the association of treatment lipids with P. gingivalis lipids revealed via thin layer chromatography, suggests that the plasma membrane is a likely target of lipid antibacterial activity. Utilizing a combination of two-dimensional in-gel electrophoresis and Western blot followed by mass spectroscopy and N-terminus degradation sequencing we also show that treatment with sapienic acid induces upregulation of a set of proteins comprising a unique P. gingivalis stress response, including proteins important in fatty acid biosynthesis, metabolism and energy production, protein processing, cell adhesion and virulence. Prophylactic or therapeutic lipid treatments may be beneficial for intervention of infection by supplementing the natural immune function of endogenous lipids on mucosal surfaces.

  7. Oral mucosal lipids are antibacterial against Porphyromonas gingivalis, induce ultrastructural damage, and alter bacterial lipid and protein compositions

    PubMed Central

    Fischer, Carol L; Walters, Katherine S; Drake, David R; Dawson, Deborah V; Blanchette, Derek R; Brogden, Kim A; Wertz, Philip W

    2013-01-01

    Oral mucosal and salivary lipids exhibit potent antimicrobial activity for a variety of Gram-positive and Gram-negative bacteria; however, little is known about their spectrum of antimicrobial activity or mechanisms of action against oral bacteria. In this study, we examine the activity of two fatty acids and three sphingoid bases against Porphyromonas gingivalis, an important colonizer of the oral cavity implicated in periodontitis. Minimal inhibitory concentrations, minimal bactericidal concentrations, and kill kinetics revealed variable, but potent, activity of oral mucosal and salivary lipids against P. gingivalis, indicating that lipid structure may be an important determinant in lipid mechanisms of activity against bacteria, although specific components of bacterial membranes are also likely important. Electron micrographs showed ultrastructural damage induced by sapienic acid and phytosphingosine and confirmed disruption of the bacterial plasma membrane. This information, coupled with the association of treatment lipids with P. gingivalis lipids revealed via thin layer chromatography, suggests that the plasma membrane is a likely target of lipid antibacterial activity. Utilizing a combination of two-dimensional in-gel electrophoresis and Western blot followed by mass spectroscopy and N-terminus degradation sequencing we also show that treatment with sapienic acid induces upregulation of a set of proteins comprising a unique P. gingivalis stress response, including proteins important in fatty acid biosynthesis, metabolism and energy production, protein processing, cell adhesion and virulence. Prophylactic or therapeutic lipid treatments may be beneficial for intervention of infection by supplementing the natural immune function of endogenous lipids on mucosal surfaces. PMID:23867843

  8. Enhanced C-type lysozyme content of wood duck (Aix sponsa) egg white: an adaptation to cavity nesting?

    PubMed

    Wellman-Labadie, Olivier; Picman, Jaroslav; Hincke, Maxwell T

    2008-01-01

    Abstract Wild waterfowl species often nest in conditions where high humidity and microbial contamination may influence egg survival and quality. Albumen is traditionally regarded as the major impediment to microbial contamination of eggs, and its composition and activity may be selected by environmental pressures. Egg white protein from the eggs of wood duck (Aix sponsa), hooded merganser (Lophodytes cucullatus), Canada goose (Branta canadensis), and mute swan (Cygnus olor) was evaluated in order to compare the antimicrobial defenses of these species. Ovotransferrin and ovalbumin were identified in all species, but c-type lysozyme was present only in wood duck and hooded merganser egg white samples. Wood duck egg white showed the greatest bacterial activity as well as the highest lysozyme content. Egg white from wood duck and hooded merganser possessed greater lysozyme activity under acidic conditions, suggesting a c-type lysozyme with a pH optimum lower than that of Gallus gallus c-type lysozyme or the presence of g-type lysozyme. Ovotransferrin bacteriostatic activity appeared to be similar across the species investigated. The results suggest that lysozyme and ovotransferrin play a role in the antimicrobial defense of the avian egg. High levels of the broad-acting c-type lysozyme appear to have evolved in the albumen of the wood duck in order to ensure proper development of the embryo in the humid conditions of the cavity nest.

  9. Comparative insight into surfactants mediated amyloidogenesis of lysozyme.

    PubMed

    Chaturvedi, Sumit K; Khan, Javed M; Siddiqi, Mohammad K; Alam, Parvez; Khan, Rizwan H

    2016-02-01

    Electrostatic and hydrophobic interactions have an important role in the protein aggregation. In this study, we have investigated the effect of charge and hydrophobicity of oppositely charged surfactants i.e., anionic (AOT and SDS) and cationic (CTAB and DTAB) on hen egg white lysozyme at pH 9.0 and 13.0, respectively. We have employed various methods such as turbidity measurements, Rayleigh light scattering, ThT, Congo red and ANS dye binding assays, far-UV CD, atomic force microscopy, transmission electron and fluorescence microscopy. At lower molar ratio, both anionic and cationic surfactants promote amyloid fibril formation in lysozyme at pH 9.0 and 13.0, respectively. The aggregation was proportionally increased with respect to protein concentration and hydrophobicity of surfactant. The morphology of aggregates at both the pH was fibrillar in structure, as visualized by dye binding and microscopic imaging techniques. Initially, the interaction between surfactants and lysozyme was electrostatic and then hydrophobic as investigated by ITC. This study demonstrates the crucial role of charge and hydrophobicity during amyloid fibril formation.

  10. A zinc complex of heparan sulfate destabilises lysozyme and alters its conformation

    SciTech Connect

    Hughes, Ashley J.; Hussain, Rohanah; Cosentino, Cesare; Guerrini, Marco; Siligardi, Giuliano; Yates, Edwin A.; Rudd, Timothy R.

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer Zinc-heparan sulfate complex destabilises lysozyme, a model amyloid protein. Black-Right-Pointing-Pointer Addition of zinc, without heparan sulfate, stabilises lysozyme. Black-Right-Pointing-Pointer Heparan sulfate cation complexes provide alternative protein folding routes. -- Abstract: The naturally occurring anionic cell surface polysaccharide heparan sulfate is involved in key biological activities and is implicated in amyloid formation. Following addition of Zn-heparan sulfate, hen lysozyme, a model amyloid forming protein, resembled {beta}-rich amyloid by far UV circular dichroism (increased {beta}-sheet: +25%), with a significantly reduced melting temperature (from 68 to 58 Degree-Sign C) by fluorescence shift assay. Secondary structure stability of the Zn-heparan sulfate complex with lysozyme was also distinct from that with heparan sulfate, under stronger denaturation conditions using synchrotron radiation circular dichroism. Changing the cation associated with heparan sulfate is sufficient to alter the conformation and stability of complexes formed between heparan sulfate and lysozyme, substantially reducing the stability of the protein. Complexes of heparan sulfate and cations, such as Zn, which are abundant in the brain, may provide alternative folding routes for proteins.

  11. PAAR-Rhs proteins harbor various C-terminal toxins to diversify the antibacterial pathways of type VI secretion systems.

    PubMed

    Ma, Jiale; Sun, Min; Dong, Wenyang; Pan, Zihao; Lu, Chengping; Yao, Huochun

    2017-01-01

    The type VI secretion system (T6SS) of bacteria plays a key role in competing for specific niches by the contact-dependent killing of competitors. Recently, Rhs proteins with polymorphic C-terminal toxin-domains that inhibit or kill neighboring cells were identified. In this report, we identified a novel Rhs with an MPTase4 (Metallopeptidase-4) domain (designated as Rhs-CT1) that showed an antibacterial effect via T6SS in Escherichia coli. We managed to develop a specific strategy by matching the diagnostic domain-architecture of Rhs-CT1 (Rhs with an N-terminal PAAR-motif and a C-terminal toxin domain) for effector retrieval and discovered a series of Rhs-CTs in E. coli. Indeed, the screened Rhs-CT3 with a REase-3 (Restriction endonuclease-3) domain also mediated interbacterial antagonism. Further analysis revealed that vgrGO1 and eagR/DUF1795 (upstream of rhs-ct) were required for the delivery of Rhs-CTs, suggesting eagR as a potential T6SS chaperone. In addition to chaperoned Rhs-CTs, neighborless Rhs-CTs could be classified into a distinct family (Rhs-Nb) sharing close evolutionary relationship with T6SS2-Rhs (encoded in the T6SS2 cluster of E. coli). Notably, the Rhs-Nb-CT5 was confirmed bioinformatically and experimentally to mediate interbacterial antagonism via Hcp2B-VgrG2 module. In a further retrieval analysis, we discovered various toxin/immunity pairs in extensive bacterial species that could be systematically classified into eight referential clans, suggesting that Rhs-CTs greatly diversify the antibacterial pathways of T6SS.

  12. The role of the confined water in the dynamic crossover of hydrated lysozyme powders.

    PubMed

    Kurzweil-Segev, Y; Greenbaum Gutina, A; Popov, I; Golodnitsky, D; Feldman, Yu

    2016-04-28

    Water is of fundamental importance for life since it plays a critical role in biological systems. An organism can only function if its macromolecules and other bioactive molecules are hydrated. However, currently there is a gap in the understanding of how protein interfaces affect water's structure and properties. This work presents combined dielectric and calorimetric measurements of hydrated lysozyme powders with different levels of hydration in a broad temperature interval. We chose lysozyme as a test sample since this globular protein has a well-defined pore with an active hydrophilic center inside. Based on the dielectric and calorimetric tests it was shown that a water quasi-solution, which contains the protein residues, has a glass transition temperature at around 155 ± 3 K. The water confined in the pore of the active center of the lysozyme has its melting temperature at around 186 ± 3 K. Melting of confined water is believed to liberate the internal motions of protein macromolecules.

  13. Refolding of detergent-denatured lysozyme using β-cyclodextrin-assisted ion exchange chromatography.

    PubMed

    Zhang, Li; Zhang, Qinming; Wang, Chaozhan

    2013-03-01

    Chromatography-based protein refolding is widely used. Detergent is increasingly used for protein solubilization from inclusion bodies. Therefore, it is necessary to develop a refolding method for detergent-denatured/solubilized proteins based on liquid chromatography. In the present work, sarkosyl-denatured/dithiothreitol-reduced lysozyme was used as a model, and a refolding method based on ion exchange chromatography, assisted by β-cyclodextrin, was developed for refolding detergent-denatured proteins. Many factors affecting the refolding, such as concentration of urea, concentration of β-cyclodextrin, pH and flow rate of mobile phases, were investigated to optimize the refolding conditions for sarkosyl-denatured lysozymes. The results showed that the sarkosyl-denatured lysozyme could be successfully refolded using β-cyclodextrin-assisted ion exchange chromatography.

  14. Macroporous chitin affinity membranes for lysozyme separation.

    PubMed

    Ruckenstein, E; Zeng, X

    1997-12-20

    Macroporous chitin membranes with high, controlled porosity and good mechanical properties have been prepared using a technique developed in this laboratory based on silica particles as porogen. They were employed for the affinity separation of lysozyme. Chitin membranes (1 mm thickness) can be operated at high fluxes (>/=1.1 mL/min/cm(2)) corresponding to pressure drops >/=2 psi. Their adsorption capacity for lysozyme ( approximately 50 mg/mL membrane) is by an order of magnitude higher than that of the chitin beads employed in column separation. In a binary mixture of lysozyme and ovalbumin, the membranes showed very high selectivity towards lysozyme. The effect of some important operation parameters, such as the flow rates during loading and elution were investigated. Lysozyme of very high purity (>98%) was obtained from a mixture of lysozyme and ovalbumin, and from egg white. The results indicate that the macroporous chitin membranes can be used for the separation, purification, and recovery of lysozyme at large scale. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 610-617, 1997.

  15. Heat-Denatured Lysozyme Inactivates Murine Norovirus as a Surrogate Human Norovirus.

    PubMed

    Takahashi, Hajime; Nakazawa, Moemi; Ohshima, Chihiro; Sato, Miki; Tsuchiya, Tomoki; Takeuchi, Akira; Kunou, Masaaki; Kuda, Takashi; Kimura, Bon

    2015-07-02

    Human norovirus infects humans through the consumption of contaminated food, contact with the excrement or vomit of an infected person, and through airborne droplets that scatter the virus through the air. Being highly infectious and highly viable in the environment, inactivation of the norovirus requires a highly effective inactivating agent. In this study, we have discovered the thermal denaturing capacity of a lysozyme with known antimicrobial activity against gram-positive bacteria, as well as its inactivating effect on murine norovirus. This study is the first report on the norovirus-inactivating effects of a thermally denatured lysozyme. We observed that lysozymes heat-treated for 40 min at 100 °C caused a 4.5 log reduction in infectivity of norovirus. Transmission electron microscope analysis showed that virus particles exposed to thermally denatured lysozymes were expanded, compared to the virus before exposure. The amino acid sequence of the lysozyme was divided into three sections and the peptides of each artificially synthesised, in order to determine the region responsible for the inactivating effect. These results suggest that thermal denaturation of the lysozyme changes the protein structure, activating the region responsible for imparting an inactivating effect against the virus.

  16. [Studies on formation of aggregates from denatured lysozymes upon renaturing with size exclusion chromatography].

    PubMed

    Bian, Liujiao; Yang, Xiaoyan; Liu, Li

    2005-03-01

    In order to study the renaturation mechanism of denatured protein in denaturant solution, the renaturation and separation process for three kinds of lysozyme molecules, which were separately denatured by urea and guanidine hydrochloride in the presence of reducing agents, was studied by size exclusion chromatography. When initial lysozyme concentration in denaturant solution was more than 10 g/L, the denatured lysozyme molecules were renatured and isolated in a size exclusion chromatographic column. A refolded lysozyme intermediate, a bi-molecular aggregate, was found. This result was confirmed by non-reducing sodium dodecyl sulfate-polyacrylamide gel electrohoresis (SDS-PAGE) analysis of renatured lysozyme molecules with the dilution method. Compared with the dilution method, the amount of the bi-molecular aggregate found by size exclusion chromatography was far less than that found in the dilution method. This result shows that the process for the renaturing of denatured lysozyme molecules in solution can be well described with three-state model in the presence of reducing agents.

  17. A comparison of the physical properties of ultrasonically synthesized lysozyme- and BSA-shelled microbubbles.

    PubMed

    Vong, Fiona; Son, Younggyu; Bhuiyan, Sadia; Zhou, Meifang; Cavalieri, Francesca; Ashokkumar, Muthupandian

    2014-01-01

    Ultrasonic technique has been used for synthesising protein microspheres possessing specific physical and functional properties. Various proteins have been used as shell materials under different experimental conditions. In previous studies, thermal or chemical denaturation of the proteins was used to obtain stable bovine-serum albumin (BSA) and lysozyme microbubbles (MBs), respectively. It is ideal to establish a generic procedure to synthesise microspheres irrespective of the nature of the protein. In order to see if a generic procedure can be established, ultrasonic synthesis of lysozyme and BSA MBs was carried out under similar experimental conditions and their properties were evaluated. The size, size distribution and the stability of the MBs were significantly different for the lysozyme and BSA MBs. The size and size distribution of the lysozyme coated MBs were larger than BSA bubbles. The mechanical strength of MBs against the shear forces, generated when irradiated by high frequency ultrasound, was studied using pulsed-sonoluminescence (SL). This study indicated that lysozyme MBs were significantly more stable than BSA MBs. An increase in mechanical strength of the MBs may lead to an increase in their storage lifetime and stability against gas diffusion. Possible reasons for such observations have been discussed.

  18. Impact of a Reducing Agent on the Dynamic Surface Properties of Lysozyme Solutions.

    PubMed

    Tihonov, Michael M; Kim, Viktoria V; Noskov, Boris A

    2016-05-01

    Disulfide bond shuffling in the presence of the reducing agents dithiothreitol (DTT) or β-mercaptoethanol (BME) strongly affects the surface properties of lysozyme solutions. The addition of 0.32 mM DTT substantially alters the kinetic dependencies of the dynamic surface elasticity and surface tension relative to those of pure protein solutions. The significant increase in the dynamic surface elasticity likely relates to the cross-linking between lysozyme molecules and the formation of a dense layer of protein globules stabilized by intermolecular disulfide bonds at the liquid/gas interface. This effect differs from the previously described influence of chaotropic denaturants, such as guanidine hydrochloride (GuHCl) and urea, on the surface properties of lysozyme solutions. If both chaotropic and reducing agents are added to protein solutions simultaneously, their effects become superimposed. In the case of mixed lysozyme/GuHCl/DTT solutions, the dynamic surface elasticity near equilibrium decreases as the GuHCl concentration increases because of the gradual loosening of the cross-linked layer of protein globules but remains much higher than that of lysozyme/GuHCl solutions.

  19. Bunching effect in single-molecule T4 lysozyme nonequilibrium conformational dynamics under enzymatic reactions.

    PubMed

    Wang, Yuanmin; Lu, H Peter

    2010-05-20

    The bunching effect, implying that conformational motion times tend to bunch in a finite and narrow time window, is observed and identified to be associated with substrate-enzyme complex formation in T4 lysozyme conformational dynamics under enzymatic reactions. Using single-molecule fluorescence spectroscopy, we have probed T4 lysozyme conformational motions under the hydrolysis reaction of polysaccharide of E. coli B cell walls by monitoring the fluorescence resonant energy transfer (FRET) between a donor-acceptor probe pair tethered to T4 lysozyme domains involving open-close hinge-bending motions. On the basis of the single-molecule spectroscopic results, molecular dynamics simulation, and a random walk model analysis, multiple intermediate states have been estimated in the evolution of T4 lysozyme enzymatic reaction active complex formation (Chen, Y.; Hu, D.; Vorpagel, E. R.; Lu, H. P. Probing single-molecule T4 lysozyme conformational dynamics by intramolecular fluorescence energy transfer. J. Phys. Chem. B 2003, 107, 7947-7956). In this Article, we report progress on the analysis of the reported experimental results, and we have identified the bunching effect of the substrate-enzyme active complex formation time in T4 lysozyme enzymatic reactions. We show that the bunching effect, a dynamic behavior observed for the catalytic hinge-bending conformational motions of T4 lysozyme, is a convoluted outcome of multiple consecutive Poisson rate processes that are defined by protein functional motions under substrate-enzyme interactions; i.e., convoluted multiple Poisson rate processes give rise to the bunching effect in the enzymatic reaction dynamics. We suggest that the bunching effect is likely common in protein conformational dynamics involved in conformation-gated protein functions.

  20. Design and structural analysis of an engineered thermostable chicken lysozyme.

    PubMed Central

    Shih, P.; Kirsch, J. F.

    1995-01-01

    A hyperstable (hs) variant of chicken egg-white lysozyme with enhanced thermal (delta Tm approximately +10.5 degrees C) and chemical (delta Cm for guanidine hydrochloride denaturation = +1.3 M) stabilities relative to wild-type (WT) was constructed by combining several individual stabilizing substitutions. The free energy difference between the native and denatured states of the hs variant is 3.1 (GdnHCl, 25 degrees C) to 4.0 (differential scanning calorimetry, 74 degrees C) kcal mol-1 greater than that of WT. The specific activity of the hs variant is 2.5-fold greater than that of WT. The choice of mutations came from diverse sources: (1) The I55L/S91T core construct with delta Tm = 3.3 degrees C from WT was available from the accompanying study (Shih P, Holland DR, Kirsch JF, 1995, Protein Sci 4:2050-2062). (2) The A31V mutation was suggested by the better atomic packing in the human lysozyme structure where the Ala 31 equivalent is Leu. (3) The H15L and R114H substitutions were selected on the basis of sequence comparisons with pheasant lysozymes that are more stable than the chicken enzyme. (4) The D101S variant was identified from a screen of mutants previously prepared in this laboratory. The effects of the individual mutations on stability are cumulative and nearly additive. PMID:8535242

  1. Protein adsorption, fibroblast activity and antibacterial properties of poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) grafted with chitosan and chitooligosaccharide after immobilized with hyaluronic acid.

    PubMed

    Hu, S-G; Jou, C-H; Yang, M C

    2003-07-01

    Poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) membrane was treated with ozone and grafted with acrylic acid. The resulting membranes were further grafted with chitosan (CS) or chitooligosaccharide (COS) via esterification. Afterward hyaluronic acid (HA) was immobilized onto CS- or COS-grafting membranes. The antibacterial activity of CS and COS against Staphylococus aureus, Escherichia coli, and Pseudomonas aeruginosa was preserved after HA immobilization. Among them, CS-grafted PHBV membrane showed higher antibacterial activity than COS-grafted PHBV membrane. In addition, after CS- or COS-grafting, the L929 fibroblasts attachment and protein adsorption were improved, while the cell number was decrease. After immobilizing HA, the cell proliferation was promoted, the protein adsorption was decreased, and the cell attachment was slightly lower than CS- or COS-grafting PHBV.

  2. In Silico Characterization of the Binding Affinity of Dendrimers to Penicillin-Binding Proteins (PBPs): Can PBPs be Potential Targets for Antibacterial Dendrimers?

    PubMed

    Ahmed, Shaimaa; Vepuri, Suresh B; Ramesh, Muthusamy; Kalhapure, Rahul; Suleman, Nadia; Govender, Thirumala

    2016-04-01

    We have shown that novel silver salts of poly (propyl ether) imine (PETIM) dendron and dendrimers developed in our group exhibit preferential antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus aureus. This led us to examine whether molecular modeling methods could be used to identify the key structural design principles for a bioactive lead molecule, explore the mechanism of binding with biological targets, and explain their preferential antibacterial activity. The current article reports the conformational landscape as well as mechanism of binding of generation 1 PETIM dendron and dendrimers to penicillin-binding proteins (PBPs) in order to understand the antibacterial activity profiles of their silver salts. Molecular dynamics at different simulation protocols and conformational analysis were performed to elaborate on the conformational features of the studied dendrimers, as well as to create the initial structure for further binding studies. The results showed that for all compounds, there were no significant conformational changes due to variation in simulation conditions. Molecular docking calculations were performed to investigate the binding theme between the studied dendrimers and PBPs. Interestingly, in significant accordance with the experimental data, dendron and dendrimer with aliphatic cores were found to show higher activity against S. aureus than the dendrimer with an aromatic core. The latter showed higher activity against MRSA. The findings from this computational and molecular modeling report together with the experimental results serve as a road map toward designing more potent antibacterial dendrimers against resistant bacterial strains.

  3. A photochemically induced dynamic nuclear polarization study of denatured states of lysozyme

    SciTech Connect

    Broadhurst, R.W.; Dobson, C.M.; Hore, P.J.; Radford, S.E.; Rees, M.L. )

    1991-01-01

    Photochemically induced dynamic nuclear polarization (photo-CIDNP) techniques have been used to examine denatured states of lysozyme produced under a variety of conditions. {sup 1}H CIDNP difference spectra of lysozyme denatured thermally, by the addition of 10 M urea, or by the complete reduction of its four disulfide bonds were found to differ substantially not only from the spectrum of the native protein but also from that expected for a completely unstructured polypeptide chain. Specifically, denatured lysozyme showed a much reduced enhancement of tryptophan relative to tyrosine than did a mixture of blocked amino acids with the same composition as the intact protein. By contrast, the CIDNP spectrum of lysozyme denatured in dimethyl sulfoxide solution was found to be similar to that expected for a random coil. It is proposed that nonrandom hydrophobic interactions are present within the denatured states of lysozyme in aqueous solution and that these reduce the reactivity of tryptophan residues relative to tyrosine residues. Characterization of such interactions is likely to be of considerable significance for an understanding of the process of protein folding.

  4. Adsorption and Unfolding of Lysozyme at a Polarized Aqueous-Organic Liquid Interface.

    PubMed

    Arooj, Mahreen; Gandhi, Neha S; Kreck, Cara A; Arrigan, Damien W M; Mancera, Ricardo L

    2016-03-31

    The adsorption of proteins at the interface between two immiscible electrolyte solutions has been found to be key to their bioelectroactivity at such interfaces. Combined with interfacial complexation of organic phase anions by cationic proteins, this adsorption process may be exploited to achieve nanomolar protein detection. In this study, replica exchange molecular dynamics simulations have been performed to elucidate for the first time the molecular mechanism of adsorption and subsequent unfolding of hen egg white lysozyme at low pH at a polarized 1,2-dichloroethane/water interface. The unfolding of lysozyme was observed to occur as soon as it reaches the organic-aqueous interface, which resulted in a number of distinct orientations at the interface. In all cases, lysozyme interacted with the organic phase through regions rich in nonpolar amino acids, such that the side chains are directed toward the organic phase, whereas charged and polar residues were oriented toward the aqueous phase. By contrast, as expected, lysozyme in neat water at low pH does not exhibit significant structural changes. These findings demonstrate the key influence of the organic phase upon adsorption of lysozyme under the influence of an electric field, which results in the unfolding of its structure.

  5. Antibacterial drug treatment increases intestinal bile acid absorption via elevated levels of ileal apical sodium-dependent bile acid transporter but not organic solute transporter α protein.

    PubMed

    Miyata, Masaaki; Hayashi, Kenjiro; Yamakawa, Hiroki; Yamazoe, Yasushi; Yoshinari, Kouichi

    2015-01-01

    Antibacterial drug treatment increases the bile acid pool size and hepatic bile acid concentration through the elevation of hepatic bile acid synthesis. However, the involvement of intestinal bile acid absorption in the increased bile acid pool size remains unclear. To determine whether intestinal bile acid absorption contributes to the increased bile acid pool in mice treated with antibacterial drugs, we evaluated the levels of bile acid transporter proteins and the capacity of intestinal bile acid absorption. Ileal apical sodium-dependent bile acid transporter (ASBT) mRNA and protein levels were significantly increased in ampicillin (ABPC)-treated mice, whereas organic solute transporter α (OSTα) mRNA levels, but not protein levels, significantly decreased in mice. Similar alterations in the expression levels of bile acid transporters were observed in mice treated with bacitracin/neomycin/streptomycin. The capacity for intestinal bile acid absorption was evaluated by an in situ loop method. Increased ileal absorption of taurochenodeoxycholic acid was observed in mice treated with ABPC. These results suggest that intestinal bile acid absorption is elevated in an ASBT-dependent manner in mice treated with antibacterial drugs.

  6. Rational design of broad spectrum antibacterial activity based on a clinically relevant enoyl-acyl carrier protein (ACP) reductase inhibitor.

    PubMed

    Schiebel, Johannes; Chang, Andrew; Shah, Sonam; Lu, Yang; Liu, Li; Pan, Pan; Hirschbeck, Maria W; Tareilus, Mona; Eltschkner, Sandra; Yu, Weixuan; Cummings, Jason E; Knudson, Susan E; Bommineni, Gopal R; Walker, Stephen G; Slayden, Richard A; Sotriffer, Christoph A; Tonge, Peter J; Kisker, Caroline

    2014-06-06

    Determining the molecular basis for target selectivity is of particular importance in drug discovery. The ideal antibiotic should be active against a broad spectrum of pathogenic organisms with a minimal effect on human targets. CG400549, a Staphylococcus-specific 2-pyridone compound that inhibits the enoyl-acyl carrier protein reductase (FabI), has recently been shown to possess human efficacy for the treatment of methicillin-resistant Staphylococcus aureus infections, which constitute a serious threat to human health. In this study, we solved the structures of three different FabI homologues in complex with several pyridone inhibitors, including CG400549. Based on these structures, we rationalize the 65-fold reduced affinity of CG400549 toward Escherichia coli versus S. aureus FabI and implement concepts to improve the spectrum of antibacterial activity. The identification of different conformational states along the reaction coordinate of the enzymatic hydride transfer provides an elegant visual depiction of the relationship between catalysis and inhibition, which facilitates rational inhibitor design. Ultimately, we developed the novel 4-pyridone-based FabI inhibitor PT166 that retained favorable pharmacokinetics and efficacy in a mouse model of S. aureus infection with extended activity against Gram-negative and mycobacterial organisms.

  7. EC300: a phage-based, bacteriolysin-like protein with enhanced antibacterial activity against Enterococcus faecalis.

    PubMed

    Proença, Daniela; Leandro, Clara; Garcia, Miguel; Pimentel, Madalena; São-José, Carlos

    2015-06-01

    Bacteriophage lytic enzymes, either endolysins or virion-associated lysins, have been receiving considerable attention as potential antibacterial agents, particularly for the combat of antibiotic-resistant Gram-positive pathogens. A conclusion that easily emerges from the careful analysis of a great number of reports on the field is that the activity of phage lytic enzymes is rarely studied in conditions that support robust growth of the target bacteria. Here, we report the construction and study of a chimerical lysin, EC300, which was designed to target and kill Enterococcus faecalis in conditions supporting vigorous bacterial growth. EC300 resulted from the fusion of a predicted M23 endopeptidase domain of a virion-associated lysin to the putative cell wall binding domain of a previously characterized amidase endolysin, both produced by the E. faecalis phage F170/08. This bacteriolysin-like protein exhibited a clear enhanced lytic activity over the parental endolysin when both were assayed in a rich bacterial growth medium. We demonstrate the killing efficacy of EC300 against growing cells of a panel of typed E. faecalis clinical strains with high level of antibiotic resistance. The possible reasons for the marked difference between the lytic performance of EC300 and that of the amidase are discussed.

  8. The Natural Antimicrobial Enzyme Lysozyme is Up-Regulated in Gastrointestinal Inflammatory Conditions

    PubMed Central

    Rubio, Carlos A.

    2014-01-01

    The cells that line the mucosa of the human gastrointestinal tract (GI, that is, oral cavity, oesophagus, stomach, small intestine, large intestine, and rectum) are constantly challenged by adverse micro-environmental factors, such as different pH, enzymes, and bacterial flora. With exception of the oral cavity, these microenvironments also contain remnant cocktails of secreted enzymes and bacteria from upper organs along the tract. The density of the GI bacteria varies, from 103/mL near the gastric outlet, to 1010/mL at the ileocecal valve, to 1011 to 1012/mL in the colon. The total microbial population (ca. 1014) exceeds the total number of cells in the tract. It is, therefore, remarkable that despite the prima facie inauspicious mixture of harmful secretions and bacteria, the normal GI mucosa retains a healthy state of cell renewal. To counteract the hostile microenvironment, the GI epithelia react by speeding cell exfoliation (the GI mucosa has a turnover time of two to three days), by increasing peristalsis, by eliminating bacteria through secretion of plasma cell-immunoglobulins and by increasing production of natural antibacterial compounds, such as defensin-5 and lysozyme. Only recently, lysozyme was found up-regulated in Barrett’s oesophagitis, chronic gastritis, gluten-induced atrophic duodenitis (coeliac disease), collagenous colitis, lymphocytic colitis, and Crohn’s colitis. This up-regulation is a response directed to the special types of bacteria recently detected in these diseases. The aim of lysozyme up-regulation is to protect individual mucosal segments to chronic inflammation. The molecular mechanisms connected to the crosstalk between the intraluminal bacterial flora and the production of lysozyme released by the GI mucosae, are discussed. Bacterial resistance continues to exhaust our supply of commercial antibiotics. The potential use of lysozyme to treat infectious diseases is receiving much attention. PMID:25437608

  9. [Therapeutic properties of proteins and peptides from colostrum and milk].

    PubMed

    Zimecki, Michał; Artym, Jolanta

    2005-01-01

    Colostrum and milk are rich in proteins and peptides which play a crucial role in innate immunity when transferred to the offspring and may accelerate maturation of the immune system in neonates. The immunotropic properties of these proteins prompted investigators research their potential application in prevention and therapy. Lactoferrin (LF) exhibits antibacterial, antifungal, antiviral, antiparasitice, and antitumoral activities. It is protective with regard to intestinal epithelium, promotes bone growth, and accelerates the recovery of immune system function in immunocompromised animals. LF was tried in the treatment of hepatitis C infection and the intestinal form of graft-versus-host disease (GvHD). A proline-rich polypeptide (PRP) demonstrated a variety of immunotropic functions, including the promotion of T-cell maturation and inhibition of autoimmune disorders. PRP, in the form of chewable tablets (Colostrinin) was recently found to improve or stabilize the health status of Alzheimer's disease patients. Casein and casein-derived peptides showed protective activities in enamel demineralization and as caries-preventing agents. The protein hydrolyzates were also protective in diabetic animals, reduced tumor growth, had antihypertensive activity and diminished colicky symptoms in infants. Glycomacropeptide (GMP), a peptide derived from kappa-casein, exhibited various antibacterial and antithrombotic activities. Alpha-lactalbumin (LA) demonstrated antiviral, antitumoral and anti-stress properties. LA-enriched diets were anxiolytic, lowered blood pressure in rats, prevented diarrhea, and led to a better weight gain in malnourished children. HAMLET, a complex of LA and oleic acid, was effective in patients with cutaneous papillomas. Lysozyme found application in infant formulas, the treatment of periodentitis, and the prevention of tooth decay. Milk enriched in lysozyme was used in feeding premature infants suffering from concomitant diseases. Interesting

  10. Solubility and binding properties of PEGylated lysozyme derivatives with increasing molecular weight on hydrophobic-interaction chromatographic resins.

    PubMed

    Müller, Egbert; Josic, Djuro; Schröder, Tim; Moosmann, Anna

    2010-07-09

    Dynamic binding capacities and resolution of PEGylated lysozyme derivatives with varying molecular weights of poly (ethylene) glycol (PEG) with 5 kDa, 10 kDa and 30 kDa for HIC resins and columns are presented. To find the optimal range for the operating conditions, solubility studies were performed by high-throughput analyses in a 96-well plate format, and optimal salt concentrations and pH values were determined. The solubility of PEG-proteins was strongly influenced by the length of the PEG moiety. Large differences in the solubilities of PEGylated lysozymes in two different salts, ammonium sulfate and sodium chloride were found. Solubility of PEGylated lysozyme derivatives in ammonium sulfate decreases with increased length of attached PEG chains. In sodium chloride all PEGylated lysozyme derivatives are fully soluble in a concentration range between 0.1 mg protein/ml and 10 mg protein/ml. The binding capacities for PEGylated lysozyme to HIC resins are dependent on the salt type and molecular weight of the PEG polymer. In both salt solutions, ammonium sulfate and sodium chloride, the highest binding capacity of the resin was found for 5 kDa PEGylated lysozyme. For both native lysozyme and 30 kDa mono-PEGylated lysozyme the binding capacities were lower. In separation experiments on a TSKgel Butyl-NPR hydrophobic-interaction column with ammonium sulfate as mobile phase, the elution order was: native lysozyme, 5 kDa mono-PEGylated lysozyme and oligo-PEGylated lysozyme. This elution order was found to be reversed when sodium chloride was used. Furthermore, the resolution of the three mono-PEGylated forms was not possible with this column and ammonium sulfate as mobile phase. In 4 M sodium chloride a resolution of all PEGylated lysozyme forms was achieved. A tentative explanation for these phenomena can be the increased solvation of the PEG polymers in sodium chloride which changes the usual attractive hydrophobic forces in ammonium sulfate to more repulsive

  11. Single-Molecule Measurements of T4 Lysozyme using Carbon Nanotube Electronic Circuits

    NASA Astrophysics Data System (ADS)

    Sims, Patrick Craig

    Because of their unique electronic and chemical properties, single-walled carbon nanotubes (SWNTs) are attractive candidates for label-free, single-molecule sensing and detection applications. In this work, a field-effect transistor (FET) architecture comprised of an individual SWNT is used to transduce the conformational motion of a single T4 lysozyme protein, conjugated to the SWNT side wall, into a corresponding electrical current signal. The SWNTs are grown using chemical vapor deposition, and metal electrical contacts are formed using electron beam evaporation. Using N-(1-Pyrene)maleimide, the protein is conjugated to the SWNT side wall. After conjugation, the sensing area of the device is submerged in an electrolyte solution, and the source-drain current is measured while applying an electrolyte-gate. Analysis of the signal provided single-molecule resolution of the dynamical activity of lysozyme as it hydrolyzes macromolecular peptidoglycan, a component of bacterial cell walls. This analysis revealed seven different independent time scales that govern the activity of lysozyme, the pH dependence of these time scales, and a lower limit on the number rate-limiting steps in lysozyme's hinge opening and closing motions. Furthermore, the signals elucidated differences in how lysozyme traverses and catalyzes structurally varying peptidoglycan constructs.

  12. Affinity adsorption of lysozyme on a macroligand prepared with Cibacron Blue 3GA attached to yeast cells.

    PubMed

    del Pilar Ferraris, María; Barrera, Guillermo I; Padilla, A Pérez; Rodríguez, Jorge A

    2011-09-15

    The objective of this study was the development of affinity adsorbent particles with the appropriate characteristics to be applied in protein purification using the affinity ultrafiltration method. To prepare affinity macroligands Cibacron Blue 3GA, as a ligand molecule, was immobilized by covalent bonding onto yeast cell walls, the support material or matrix. The maximum attachment of the ligand to the matrix was 212 μmol/g (ligand dry weight/yeast dry weight). Lysozyme was selected as the protein model for the adsorption studies. Its adsorption onto the matrix without ligand and matrix with attached ligand were investigated batch-wise. The adsorption equilibrium isotherms appeared to follow a typical Langmuir isotherm. The maximum adsorption capacity (q(m)) of the Cell-Cibacron macroligand for lysozyme was 110 mg/ml of wet macroligand. The adsorbent was also employed for the separation of lysozyme from hen egg white. High purity lysozyme was obtained.

  13. Activity and conformation of lysozyme in molecular solvents, protic ionic liquids (PILs) and salt-water systems.

    PubMed

    Wijaya, Emmy C; Separovic, Frances; Drummond, Calum J; Greaves, Tamar L

    2016-09-21

    Improving protein stabilisation is important for the further development of many applications in the pharmaceutical, specialty chemical, consumer product and agricultural sectors. However, protein stabilization is highly dependent on the solvent environment and, hence, it is very complex to tailor protein-solvent combinations for stable protein maintenance. Understanding solvent features that govern protein stabilization will enable selection or design of suitable media with favourable solution environments to retain protein native conformation. In this work the structural conformation and activity of lysozyme in 29 solvent systems were investigated to determine the role of various solvent features on the stability of the enzyme. The solvent systems consisted of 19 low molecular weight polar solvents and 4 protic ionic liquids (PILs), both at different water content levels, and 6 aqueous salt solutions. Small angle X-ray scattering, Fourier transform infrared spectroscopy and UV-vis spectroscopy were used to investigate the tertiary and secondary structure of lysozyme along with the corresponding activity in various solvation systems. At low non-aqueous solvent concentrations (high water content), the presence of solvents and salts generally maintained lysozyme in its native structure and enhanced its activity. Due to the presence of a net surface charge on lysozyme, electrostatic interactions in PIL-water systems and salt solutions enhanced lysozyme activity more than the specific hydrogen-bond interactions present in non-ionic molecular solvents. At higher solvent concentrations (lower water content), solvents with a propensity to exhibit the solvophobic effect, analogous to the hydrophobic effect in water, retained lysozyme native conformation and activity. This solvophobic effect was observed particularly for solvents which contained hydroxyl moieties. Preferential solvophobic effects along with bulky chemical structures were postulated to result in less

  14. Design, synthesis and antibacterial activity of cinnamaldehyde derivatives as inhibitors of the bacterial cell division protein FtsZ.

    PubMed

    Li, Xin; Sheng, Juzheng; Huang, Guihua; Ma, Ruixin; Yin, Fengxin; Song, Di; Zhao, Can; Ma, Shutao

    2015-06-05

    In an attempt to discover potential antibacterial agents against the increasing bacterial resistance, novel cinnamaldehyde derivatives as FtsZ inhibitors were designed, synthesized and evaluated for their antibacterial activity against nine significant pathogens using broth microdilution method, and their cell division inhibitory activity against four representative strains. In the in vitro antibacterial activity, the newly synthesized compounds generally displayed better efficacy against Staphylococcus aureus ATCC25923 than the others. In particular, compounds 3, 8 and 10 exerted superior or comparable activity to all the reference drugs. In the cell division inhibitory activity, all the compounds showed the same trend as their in vitro antibacterial activity, exhibiting better activity against S. aureus ATCC25923 than the other strains. Additionally, compounds 3, 6, 7 and 8 displayed potent cell division inhibitory activity with an MIC value of below 1 μg/mL, over 256-fold better than all the reference drugs.

  15. Molecular Insight into Human Lysozyme and Its Ability to Form Amyloid Fibrils in High Concentrations of Sodium Dodecyl Sulfate: A View from Molecular Dynamics Simulations

    PubMed Central

    Jafari, Majid; Mehrnejad, Faramarz

    2016-01-01

    Changes in the tertiary structure of proteins and the resultant fibrillary aggregation could result in fatal heredity diseases, such as lysozyme systemic amyloidosis. Human lysozyme is a globular protein with antimicrobial properties with tendencies to fibrillate and hence is known as a fibril-forming protein. Therefore, its behavior under different ambient conditions is of great importance. In this study, we conducted two 500000 ps molecular dynamics (MD) simulations of human lysozyme in sodium dodecyl sulfate (SDS) at two ambient temperatures. To achieve comparative results, we also performed two 500000 ps human lysozyme MD simulations in pure water as controls. The aim of this study was to provide further molecular insight into all interactions in the lysozyme-SDS complexes and to provide a perspective on the ability of human lysozyme to form amyloid fibrils in the presence of SDS surfactant molecules. SDS, which is an anionic detergent, contains a hydrophobic tail with 12 carbon atoms and a negatively charged head group. The SDS surfactant is known to be a stabilizer for helical structures above the critical micelle concentration (CMC) [1]. During the 500000 ps MD simulations, the helical structures were maintained by the SDS surfactant above its CMC at 300 K, while at 370 K, human lysozyme lost most of its helices and gained β-sheets. Therefore, we suggest that future studies investigate the β-amyloid formation of human lysozyme at SDS concentrations above the CMC and at high temperatures. PMID:27768744

  16. Synthesis of hydrophobic nanoparticles for real-time lysozyme detection using surface plasmon resonance sensor.

    PubMed

    Saylan, Yeşeren; Yılmaz, Fatma; Derazshamshir, Ali; Yılmaz, Erkut; Denizli, Adil

    2017-03-21

    Diagnostic biomarkers such as proteins and enzymes are generally hard to detect because of the low abundance in biological fluids. To solve this problem, the advantages of surface plasmon resonance (SPR) and nanomaterial technologies have been combined. The SPR sensors are easy to prepare, no requirement of labelling and can be detected in real time. In addition, they have high specificity and sensitivity with low cost. The nanomaterials have also crucial functions such as efficiency improvement, selectivity, and sensitivity of the detection systems. In this report, an SPR-based sensor is developed to detect lysozyme with hydrophobic poly (N-methacryloyl-(L)-phenylalanine) (PMAPA) nanoparticles. The SPR sensor was first characterized by attenuated total reflection-Fourier transform infrared, atomic force microscope, and water contact angle measurements and performed with aqueous lysozyme solutions. Various concentrations of lysozyme solution were used to calculate kinetic and affinity coefficients. The equilibrium and adsorption isotherm models of interactions between lysozyme solutions and SPR sensor were determined and the maximum reflection, association, and dissociation constants were calculated by Langmuir model as 4.87, 0.019 nM(-1) , and 54 nM, respectively. The selectivity studies of SPR sensor were investigated with competitive agents, hemoglobin, and myoglobin. Also, the SPR sensor was used four times in adsorption/desorption/recovery cycles and results showed that, the combination of optical SPR sensor with hydrophobic ionizable PMAPA nanoparticles in one mode enabled the detection of lysozyme molecule with high accuracy, good sensivity, real-time, label-free, and a low-detection limit of 0.66 nM from lysozyme solutions. Lysozyme detection in a real sample was performed by using chicken egg white to evaluate interfering molecules present in the medium.

  17. Lysozyme stability and amyloid fibrillization dependence on Hofmeister anions in acidic pH.

    PubMed

    Poniková, Slavomíra; Antošová, Andrea; Demjén, Erna; Sedláková, Dagmar; Marek, Jozef; Varhač, Rastislav; Gažová, Zuzana; Sedlák, Erik

    2015-09-01

    We have explored an effect of Hofmeister anions, Na2SO4, NaCl, NaBr, NaNO3, NaSCN and NaClO4, on stability and amyloid fibrillization of hen egg white lysozyme at pH 2.7. The stability of the protein was analyzed by differential scanning calorimetry. The Hofmeister effect of the anions was assessed by the parameter dT trs/d[anion] (T trs, transition temperature). We show that dT trs/d[anion] correlates with anion surface tension effects and anion partition coefficients indicating direct interactions between anions and lysozyme. The kinetic of amyloid fibrillization of lysozyme was followed by Thioflavin T (ThT) fluorescence. Negative correlation between dT trs/d[anion] and the nucleation rate of fibrillization in the presence of monovalent anions indicates specific effect of anions on fibrillization rate of lysozyme. The efficiency of monovalent anions to accelerate fibrillization correlates with inverse Hofmeister series. The far-UV circular dichroism spectroscopy and atomic force microscopy findings show that conformational properties of fibrils depend on fibrillization rate. In the presence of sodium chloride, lysozyme forms typical fibrils with elongated structure and with the secondary structure of the β-sheet. On the other hand, in the presence of both chaotropic perchlorate and kosmotropic sulfate anions, the fibrils form clusters with secondary structure of β-turn. Moreover, the acceleration of fibril formation is accompanied by decreased amount of the formed fibrils as indicated by ThT fluorescence. Taken together, our study shows Hofmeister effect of monovalent anions on: (1) lysozyme stability; (2) ability to accelerate nucleation phase of lysozyme fibrillization; (3) amount, and (4) conformational properties of the formed fibrils.

  18. Chondroitin sulfate is involved in lysosomal transport of lysozyme in U937 cells.

    PubMed

    Lemansky, P; Hasilik, A

    2001-01-01

    Human promonocytes U937 synthesize lysozyme and retain approximately one third of it within lysosomes. Lysozyme is not glycosylated; thus, it cannot be subject to mannose-6-phosphate-dependent targeting to lysosomes. It is a basic protein with a pI of 10.5 and is known to interact with negatively charged macromolecules like proteoglycans. Therefore, we examined whether the latter are involved in the lysosomal targeting of lysozyme in U937 cells. We partially diminished the electronegative charge of newly synthesized proteoglycans by inhibiting their sulfation with chlorate. This increased the rate of secretion of lysozyme. Upon treatment of U937 cells with phorbol esters, the rate of secretion of lysozyme was increased to more than 90%. This coincided with an almost complete redistribution of a [(35)S]sulfate bearing proteoglycan to the secretory pathway. After a brief pulse with [(35)S]sulfate in the control, 80% of the [(35)S]sulfate-bearing proteoglycan was retained within the cells, whereas in the treated cells this proportion was decreased to 13%. The secreted proteoglycan was sensitive to chondroitinase ABC and bound to immobilized lysozyme. This interaction was disrupted by 50-300 mM NaCl. The intracellularly retained proteoglycan was degraded with a half-life of 50-60 minutes and seemed to be directed to lysosomes because in the presence of NH(4)Cl the degradation was strongly inhibited. Our results suggest that the proteoglycan is involved in lysosomal targeting of lysozyme in U937 cells.

  19. X-ray studies of water in crystals of lysozyme.

    PubMed

    Blake, C C; Pulford, W C; Artymiuk, P J

    1983-07-05

    The structure of the water in crystals of human and tortoise egg-white lysozyme, which contain about 350 and about 650 water molecules per protein molecule, respectively, has been studied by X-ray refinement at high resolution. In the crystals, 60 to 80% of the total water is represented by featureless electron density filling the crystal interstices, which can be modelled to a first approximation by a single-valued, smoothed electron density continuum. The number of ordered water molecules detected is 140 for human and 128 for tortoise. These ordered water molecules are either hydrogen-bonded to protein polar groups, or hydrogen-bonded to other bound water molecules, to form a single layer around the protein molecules. Estimates of the proportion of the protein surface covered by ordered water molecules have been obtained by contact area calculations, giving a lower limit of approximately 45%, an upper limit of approximately 85% and a "best" estimate of approximately 75%. Examination of the structure of the ordered water layer shows that it is probably not any other single regular structure, and suggests that there is a local ordering controlled by the nature of the protein surface. Nearly all exposed protein polar atoms interact with ordered water molecules with, on average, protein oxygen atoms interacting with twice as many water molecules as protein nitrogen atoms. Analysis of the relation of the B-factors of the bound water molecules to the B-factors of the protein atoms to which they are bound, suggests that the 33 to 35 water molecules that make multiple hydrogen bonds with the lysozyme molecules are strongly bound, and that the 95 to 105 waters that make single hydrogen bonds to the protein or other bound water molecules are more weakly bound. Comparison of the location of the bound water molecules in the two lysozymes shows that most of the multiply bound water molecules occupy similar binding sites, suggesting that crystal packing or the presence of salt

  20. Incorporation of impurity to a tetragonal lysozyme crystal

    NASA Astrophysics Data System (ADS)

    Kurihara, Kazuo; Miyashita, Satoru; Sazaki, Gen; Nakada, Toshitaka; Durbin, Stephen D.; Komatsu, Hiroshi; Ohba, Tetsuhiko; Ohki, Kazuo

    1999-01-01

    Concentration of a phosphor-labeled impurity (ovalbumin) incorporated into protein (hen egg white lysozyme) crystals during growth was measured by fluorescence.This technique enabled us to measure the local impurity concentration in a crystal quantitatively. Impurity concentration increased with growth rate, which could not be explained by two conventional models (equilibrium adsorption model and Burton-Prim-Slichter model); a modified model is proposed. Impurity concentration also increased with the pH of the solution. This result is discussed considering the electrostatic interaction between the impurity and the crystallizing species.

  1. [Fluorescent energy transfer study of lysozyme complexes with liposomes].

    PubMed

    Gorbenko, G P

    1999-01-01

    The method of radiationless energy transfer was used to study the structure of lysozyme complexes with liposomes composed of phosphatidylcholine and diphosphatidylglycerol (4:3, mol:mol). 4-(n-Dimethylaminostyryl)-1-methylpyridinium n-toluenesulfonate, 4-(n-dimethylaminostyryl)-1-hexylpyridinium n-toluenesulfonate, 4-(n-dimethylaminostyryl)-1-dodecylpyridinium n-toluenesulfonate, and 3-metoxybenzanthrone were used as donors, and nile blue and rhodamine 6G, as acceptors. An increase in the surface area of model membranes upon binging of the protein to lipid bilayer was found.

  2. Refolding of denatured/reduced lysozyme at high concentrations by artificial molecular chaperone-ion exchange chromatography.

    PubMed

    Wang, Chaozhan; Zhang, Qinming; Cheng, Yan; Wang, Lili

    2010-01-01

    Development of high efficiency and low cost protein refolding methods is a highlighted research focus in biotechnology. Artificial molecular chaperone (AMC) and protein folding liquid chromatography (PFLC) are two attractive refolding methods developed in recent years. In the present work, AMC and one branch of PFLC, ion exchange chromatography (IEC), are integrated to form a new refolding method, artificial molecular chaperone-ion exchange chromatography (AMC-IEC). This new method is applied to the refolding of a widely used model protein, urea-denatured/dithiothreitol-reduced lysozyme. Many factors influencing the refolding of lysozyme, such as urea concentration, beta-cyclodextrin concentration, molar ratio of detergent to protein, mobile phase flow rate, and type of detergent, were investigated, respectively, to optimize the conditions for lysozyme refolding by AMC-IEC. Compared with normal IEC refolding method, the activity recoveries of lysozyme obtained by AMC-IEC were much higher in the investigated range of initial protein concentrations. Moreover, the activity recoveries obtained by using this newly developed refolding method were still quite high for denatured/reduced lysozyme at high initial concentrations. When the initial protein concentration was 200 mg mL(-1), the activity recovery was over 60%. In addition, the lifetime of the chromatographic column during AMC-IEC was much longer than that during protein refolding by normal IEC. Therefore, AMC-IEC is a high efficient and low cost protein refolding method.

  3. Expression, purification, and characterization of the recombinant calcium-binding equine lysozyme secreted by the filamentous fungus Aspergillus niger: comparisons with the production of hen and human lysozymes.

    PubMed

    Spencer, A; Morozov-Roche, L A; Noppe, W; MacKenzie, D A; Jeenes, D J; Joniau, M; Dobson, C M; Archer, D B

    1999-06-01

    Equine lysozyme (EqL) has been expressed from a synthetic gene and secreted from a heterologous host, the filamentous fungus Aspergillus niger. By including 100 mM Ca2+ in the growth medium, secreted yields of more than 50 mg/liter could be achieved using polyvinylpyrrolidone (PVP) complete medium. In a soya medium yields of up to 150 mg/liter were achieved. The production of recombinant human lysozyme (HuL) from A. niger with yields of over 40 mg/liter was also achieved using PVP medium. Addition of Ca2+ to the growth medium reduced the yield of both HuL and hen egg white lysozyme (HEWL). Sequence differences between the three lysozymes, EqL, HuL, and HEWL, resulted in different susceptibilities to cleavage by A. niger proteases. An improved procedure for the purification of EqL and HuL from A. niger allowed separation of the proteins from pigments produced by the fungus. Detailed spectroscopic analysis, including 2D 1H NMR, for recombinant EqL and recombinant HuL confirm that both proteins possess their native structure and are purified to homogeneity.

  4. Insights into Kinetics of Agitation-Induced Aggregation of Hen Lysozyme under Heat and Acidic Conditions from Various Spectroscopic Methods

    PubMed Central

    Chaari, Ali; Fahy, Christine; Chevillot-Biraud, Alexandre; Rholam, Mohamed

    2015-01-01

    Protein misfolding and amyloid formation are an underlying pathological hallmark in a number of prevalent diseases of protein aggregation ranging from Alzheimer’s and Parkinson’s diseases to systemic lysozyme amyloidosis. In this context, we have used complementary spectroscopic methods to undertake a systematic study of the self-assembly of hen egg-white lysozyme under agitation during a prolonged heating in acidic pH. The kinetics of lysozyme aggregation, monitored by Thioflavin T fluorescence, dynamic light scattering and the quenching of tryptophan fluorescence by acrylamide, is described by a sigmoid curve typical of a nucleation-dependent polymerization process. Nevertheless, we observe significant differences between the values deduced for the kinetic parameters (lag time and aggregation rate). The fibrillation process of lysozyme, as assessed by the attenuated total reflection-Fourier transform infrared spectroscopy, is accompanied by an increase in the β-sheet conformation at the expense of the α-helical conformation but the time-dependent variation of the content of these secondary structures does not evolve as a gradual transition. Moreover, the tryptophan fluorescence-monitored kinetics of lysozyme aggregation is described by three phases in which the temporal decrease of the tryptophan fluorescence quantum yield is of quasilinear nature. Finally, the generated lysozyme fibrils exhibit a typical amyloid morphology with various lengths (observed by atomic force microscopy) and contain exclusively the full-length protein (analyzed by highly performance liquid chromatography). Compared to the data obtained by other groups for the formation of lysozyme fibrils in acidic pH without agitation, this work provides new insights into the structural changes (local, secondary, oligomeric/fibrillar structures) undergone by the lysozyme during the agitation-induced formation of fibrils. PMID:26571264

  5. Crystallization, data collection and phasing of two digestive lysozymes from Musca domestica

    SciTech Connect

    Marana, S. R.; Cançado, F. C.; Valério, A. A.; Ferreira, C.; Terra, W. R.; Barbosa, J. A. R. G.

    2006-08-01

    The digestive lysozymes 1 and 2 from M. domestica were crystallized by vapour diffusion. The crystallographic data were processed to a maximum resolution of 1.9 Å in both cases. Lysozymes are mostly known for their defensive role against bacteria, but in several animals lysozymes have a digestive function. Here, the initial crystallographic characterization of two digestive lysozymes from Musca domestica are presented. The proteins were crystallized using the sitting-drop vapour-diffusion method in the presence of ammonium sulfate or PEG/2-propanol as the precipitant. X-ray diffraction data were collected to a maximum resolution of 1.9 Å using synchrotron radiation. The lysozyme 1 and 2 crystals belong to the monoclinic space group P2{sub 1} (unit-cell parameters a = 36.52, b = 79.44, c = 45.20 Å, β = 102.97°) and the orthorhombic space group P2{sub 1}2{sub 1}2 (unit-cell parameters a = 73.90, b = 96.40, c = 33.27 Å), respectively. The crystal structures were solved by molecular replacement and structure refinement is in progress.

  6. Analytical and preparative separation of PEGylated lysozyme for the characterization of chromatography media.

    PubMed

    Moosmann, Anna; Christel, Jessica; Boettinger, Heiner; Mueller, Egbert

    2010-01-08

    The effect of PEGylation on cation exchange chromatography was studied with poly(ethylene glycol) of different chain lengths (5kDa, 10kDa and 30kDa) using lysozyme as a model system. A stable binding via reduction of a Schiff base was formed during random PEGylation on lysine residues with methoxy-PEG-aldehyde. A purification method for PEGylated proteins using cation exchange chromatography was developed, and different isoforms of mono-PEGylated lysozyme were isolated. TSKgel SP-5PW and Toyopearl GigaCap S-650M showed the best performance of all tested cation exchange resins, and the separation of PEGylated lysozyme could be also scaled up to semi-preparative level. Size-exclusion chromatography, SDS-PAGE and MALDI-TOF mass spectrometry were used for analysis. Separated mono-PEGylated lysozyme of different sizes was used to determine dynamic binding capacities (DBC) and selectivity of cation exchange chromatography resins. An optimization of binding conditions resulted in a more than 20-fold increase of DBC for Toyopearl GigaCap S-650M with 30kDa mono-PEGylated lysozyme.

  7. Binding and Inhibitory Effect of the Dyes Amaranth and Tartrazine on Amyloid Fibrillation in Lysozyme.

    PubMed

    Basu, Anirban; Suresh Kumar, Gopinatha

    2017-02-16

    Interaction of two food colorant dyes, amaranth and tartrazine, with lysozyme was studied employing multiple biophysical techniques. The dyes exhibited hypochromic changes in the presence of lysozyme. The intrinsic fluorescence of lysozyme was quenched by both dyes; amaranth was a more efficient quencher than tartrazine. The equilibrium constant of amaranth was higher than that of tartarzine. From FRET analysis, the binding distances for amaranth and tartrazine were calculated to be 4.51 and 3.93 nm, respectively. The binding was found to be dominated by non-polyelectrolytic forces. Both dyes induced alterations in the microenvironment surrounding the tryptophan and tyrosine residues of the protein, with the alterations being comparatively higher for the tryptophans than the tyrosines. The interaction caused significant loss in the helicity of lysozyme, the change being higher with amaranth. The binding of both dyes was exothermic. The binding of amaranth was enthalpy driven, while that of tartrazine was predominantly entropy driven. Amaranth delayed lysozyme fibrillation at 25 μM, while tartrazine had no effect even at 100 μM. Nevertheless, both dyes had a significant inhibitory effect on fibrillogenesis. The present study explores the potential antiamyloidogenic property of these azo dyes used as food colorants.

  8. Cyclodextrin/poly(ethylene glycol) polypseudorotaxane hydrogels as a promising sustained-release system for lysozyme.

    PubMed

    Higashi, Taishi; Tajima, Anna; Motoyama, Keiichi; Arima, Hidetoshi

    2012-08-01

    In this study, to clarify the utility of polypseudorotaxane (PPRX) hydrogels composed of poly(ethylene glycol) (PEG) and α- or γ-cyclodextrin (α- or γ-CyD) as a sustained-release system for protein drugs, we prepared CyD PPRX hydrogels including lysozyme, and then the release profiles of lysozyme from these hydrogels and the release mechanisms were investigated. The α- and γ-CyD formed PPRX hydrogels by threading onto one PEG chain and two PEG chains, respectively. The formation of α- and γ-CyD PPRX hydrogels including lysozyme was based on physical cross-linking arisen from their columnar structures. The in vitro release rates of lysozyme were markedly decreased by the encapsulation into CyD PPRX hydrogels. In addition, when release data were plotted according to Korsmeyer-Peppas model, the exponent values (n) in the α- and γ-CyD systems had no statistically significant difference, suggesting that these release mechanisms were almost same. In conclusion, these results suggest that α- and γ-CyD PPRX hydrogels possess the potential as a sustained-release system for lysozyme.

  9. Hydrogen exchange of lysozyme powders. Hydration dependence of internal motions.

    PubMed

    Schinkel, J E; Downer, N W; Rupley, J A

    1985-01-15

    The rate of exchange of the labile hydrogens of lysozyme was measured by out-exchange of tritium from the protein in solution and from powder samples of varied hydration level, for pH 2, 3, 5, 7, and 10 at 25 degrees C. The dependence of exchange of powder samples on the level of hydration was the same for all pHs. Exchange increased strongly with increased hydration until reaching a rate of exchange that is constant above 0.15 g of H2O/g of protein (120 mol of H2O/mol of protein). This hydration level corresponds to coverage of less than half the protein surface with a monolayer of water. No additional hydrogen exchange was observed for protein powders with higher water content. Considered in conjunction with other lysozyme hydration data [Rupley, J. A., Gratton, E., & Careri, G. (1983) Trends Biochem. Sci. (Pers. Ed.) 8, 18-22], this observation indicates that internal protein dynamics are not strongly coupled to surface properties. The use of powder samples offers control of water activity through regulation of water vapor pressure. The dependence of the exchange rate on water activity was about fourth order. The order was pH independent and was constant from 114 to 8 mol of hydrogen remaining unexchanged/mol of lysozyme. These results indicate that the rate-determining step for protein hydrogen exchange is similar for all backbone amides and involves few water molecules. Powder samples were hydrated either by isopiestic equilibration, with a half-time for hydration of about 1 h, or by addition of solvent to rapidly reach final hydration. Samples hydrated slowly by isopiestic equilibration exhibited more exchange than was observed for samples of the same water content that had been hydrated rapidly by solvent addition. This difference can be explained by salt and pH effects on the nearly dry protein. Such effects would be expected to contribute more strongly during the isopiestic equilibration process. Solution hydrogen exchange measurements made for comparison

  10. Identification and functional characterization of a novel monotreme- specific antibacterial protein expressed during lactation.

    PubMed

    Bisana, Swathi; Kumar, Satish; Rismiller, Peggy; Nicol, Stewart C; Lefèvre, Christophe; Nicholas, Kevin R; Sharp, Julie A

    2013-01-01

    Monotremes are the only oviparous mammals and exhibit a fascinating combination of reptilian and mammalian characters. They represent a component of synapsidal reproduction by laying shelled eggs which are incubated outside the mother's body. This is accompanied by a prototherian lactation process, marking them as representatives of early mammals. The only extant monotremes are the platypus, and the short- and long- beaked echidnas, and their distributions are limited to Australia and New Guinea. Apart for a short weaning period, milk is the sole source of nutrition and protection for the hatchlings which are altricial and immunologically naive. The duration of lactation in these mammals is prolonged relative to the gestational length and period of incubation of eggs. Much of the development of monotreme young occurs in the non-sterile ex-utero environment. Therefore the role of milk in the growth, development and disease protection of the young is of significant interest. By sequencing the cDNA of cells harvested from monotreme milk, we have identified a novel monotreme- specific transcript, and the corresponding gene was designated as the EchAMP. The expression profile of this gene in various tissues revealed that it is highly expressed in milk cells. The peptides corresponding to the EchAMP protein have been identified in a sample of echidna milk In silico analysis indicated putative antimicrobial potential for the cognate protein of EchAMP. This was further confirmed by in vitro assays using a host of bacteria. Interestingly, EchAMP did not display any activity against a commensal gut floral species. These results support the hypothesis of enhancement of survival of the young by antimicrobial bioactives of mammary gland origin and thus emphasize the protective, non- nutritional role of milk in mammals.

  11. Expression, Purification, and Characteristic of Tibetan Sheep Breast Lysozyme Using Pichia pastoris Expression System

    PubMed Central

    Li, Jianbo; Jiang, Mingfeng; Wang, Yong

    2014-01-01

    A lysozyme gene from breast of Tibetan sheep was successfully expressed by secretion using a-factor signal sequence in the methylotrophic yeast, Pichia pastoris GS115. An expression yield and specific activity greater than 500 mg/L and 4,000 U/mg was obtained. Results at optimal pH and temperature showed recombinant lysozyme has higher lytic activity at pH 6.5 and 45°C. This study demonstrates the successful expression of recombinant lysozyme using the eukaryotic host organism P. pastoris paving the way for protein engineering. Additionally, this study shows the feasibility of subsequent industrial manufacture of the enzyme with this expression system together with a high purity scheme for easy high-yield purification. PMID:25049990

  12. Lysozyme effect on structural state of model membranes as revealed by pyrene excimerization studies.

    PubMed

    Ioffe, Valeriya; Gorbenko, Galyna P

    2005-04-22

    Steady-state measurements of pyrene fluorescence in the model bilayer membranes composed of phosphatidylcholine (PC) and its mixtures with cardiolipin (CL) have been performed to gain insight into the effect of lysozyme on molecular organization of lipid bilayer. Analysis of vibronic structure of the probe emission spectra revealed no changes in transverse distribution of pyrene monomers on varying CL contents or increasing the extent of lysozyme binding to liposomes. Excimer-to-monomer fluorescence intensity ratio has been found to reduce on lysozyme association with lipids. The magnitude of this effect increased with increasing CL content from 0 to 40 mol%. These results have been interpreted as indicating decrease in the membrane free volume on formation of both electrostatic and hydrophobic protein-lipid contacts.

  13. Identification and characterization of the antimicrobial peptide corresponding to C-terminal beta-sheet domain of tenecin 1, an antibacterial protein of larvae of Tenebrio molitor.

    PubMed

    Lee, K H; Hong, S Y; Oh, J E; Kwon, M; Yoon, J H; Lee, J; Lee, B L; Moon, H M

    1998-08-15

    An active fragment was identified from tenecin 1, an antibacterial protein belonging to the insect defensin family, by synthesizing the peptides corresponding to the three regions of tenecin 1. Only the fragment corresponding to the C-terminal beta-sheet domain showed activity against fungi as well as Gram-positive and Gram-negative bacteria, whereas tenecin 1, the native protein, showed activity only against Gram-positive bacteria. CD spectra indicated that each fragment in a membrane-mimetic environment might adopt a secondary structure corresponding to its region in the protein. The leakage of dye from liposomes induced by this fragment suggested that this fragment acts on the membrane of pathogens as a primary mode of action. A comparison between the structure and the activity of each fragment indicated that a net positive charge was a prerequisite factor for activity. To the best of our knowledge this is the first report in which the fragment corresponding to the beta-sheet region in antibacterial proteins, which consists of alpha-helical and beta-sheet regions, has been identified as a primary active fragment.

  14. The Effect of Ethylene Glycol, Glycine Betaine, and Urea on Lysozyme Thermal Stability

    ERIC Educational Resources Information Center

    Schwinefus, Jeffrey J.; Leslie, Elizabeth J.; Nordstrom, Anna R.

    2010-01-01

    The four-week student project described in this article is an extension of protein thermal denaturation experiments to include effects of added cosolutes ethylene glycol, glycine betaine, and urea on the unfolding of lysozyme. The transition temperatures and van't Hoff enthalpies for unfolding are evaluated for six concentrations of each cosolute,…

  15. Lysozyme Thermal Denaturation and Self-Interaction: Four Integrated Thermodynamic Experiments for the Physical Chemistry Laboratory

    ERIC Educational Resources Information Center

    Schwinefus, Jeffrey J.; Schaefle, Nathaniel J.; Muth, Gregory W.; Miessler, Gary L.; Clark, Christopher A.

    2008-01-01

    As part of an effort to infuse our physical chemistry laboratory with biologically relevant, investigative experiments, we detail four integrated thermodynamic experiments that characterize the denaturation (or unfolding) and self-interaction of hen egg white lysozyme as a function of pH and ionic strength. Students first use Protein Explorer to…

  16. Immunological function and antibacterial activity of two ferritin proteins from the freshwater pearl mussel Hyriopsis schlegelii.

    PubMed

    Sheng, J Q; Shu, Q C; Shi, J W; Wang, J H; Peng, K; Yuan, S; Hong, Y J

    2016-08-29

    Ferritin is a conserved iron-binding protein involved in host defense and cellular iron metabolism in most organisms. We investigated the expression profiles of two ferritin genes (designated HsFer-1 and HsFer-2) in the hemocytes, gonad, and hepatopancreas of Hyriopsis schlegelii, when challenged with bacteria and metal ions. HsFer gene transcription increased 1.8-7.7- and 1.9-6.1-fold in these tissues after stimulation with Staphylococcus aureus and Vibrio anguillarum, respectively. In addition, following exposure to Fe(3+), expression of HsFer-1 and HsFer-2 was elevated by 1.5-6.1- and 3.6-10.1-fold, respectively. Levels of HsFer-1 and -2 mRNA also increased significantly after treatment with Cu(2+) and Pb(2+) at certain concentrations. Moreover, recombinant HsFer-1 and -2 were able to inhibit the growth of two strains of bacteria, and the former efficiently chelated Fe(3+). From these results, we conclude that HsFer-1 and -2 may be involved in iron metabolism and immune defense by inhibiting the growth of bacteria.

  17. Glycosyltransferases and Transpeptidases/Penicillin-Binding Proteins: Valuable Targets for New Antibacterials

    PubMed Central

    Sauvage, Eric; Terrak, Mohammed

    2016-01-01

    Peptidoglycan (PG) is an essential macromolecular sacculus surrounding most bacteria. It is assembled by the glycosyltransferase (GT) and transpeptidase (TP) activities of multimodular penicillin-binding proteins (PBPs) within multiprotein complex machineries. Both activities are essential for the synthesis of a functional stress-bearing PG shell. Although good progress has been made in terms of the functional and structural understanding of GT, finding a clinically useful antibiotic against them has been challenging until now. In contrast, the TP/PBP module has been successfully targeted by β-lactam derivatives, but the extensive use of these antibiotics has selected resistant bacterial strains that employ a wide variety of mechanisms to escape the lethal action of these antibiotics. In addition to traditional β-lactams, other classes of molecules (non-β-lactams) that inhibit PBPs are now emerging, opening new perspectives for tackling the resistance problem while taking advantage of these valuable targets, for which a wealth of structural and functional knowledge has been accumulated. The overall evidence shows that PBPs are part of multiprotein machineries whose activities are modulated by cofactors. Perturbation of these systems could lead to lethal effects. Developing screening strategies to take advantage of these mechanisms could lead to new inhibitors of PG assembly. In this paper, we present a general background on the GTs and TPs/PBPs, a survey of recent issues of bacterial resistance and a review of recent works describing new inhibitors of these enzymes. PMID:27025527

  18. Glycosyltransferases and Transpeptidases/Penicillin-Binding Proteins: Valuable Targets for New Antibacterials.

    PubMed

    Sauvage, Eric; Terrak, Mohammed

    2016-02-17

    Peptidoglycan (PG) is an essential macromolecular sacculus surrounding most bacteria. It is assembled by the glycosyltransferase (GT) and transpeptidase (TP) activities of multimodular penicillin-binding proteins (PBPs) within multiprotein complex machineries. Both activities are essential for the synthesis of a functional stress-bearing PG shell. Although good progress has been made in terms of the functional and structural understanding of GT, finding a clinically useful antibiotic against them has been challenging until now. In contrast, the TP/PBP module has been successfully targeted by β-lactam derivatives, but the extensive use of these antibiotics has selected resistant bacterial strains that employ a wide variety of mechanisms to escape the lethal action of these antibiotics. In addition to traditional β-lactams, other classes of molecules (non-β-lactams) that inhibit PBPs are now emerging, opening new perspectives for tackling the resistance problem while taking advantage of these valuable targets, for which a wealth of structural and functional knowledge has been accumulated. The overall evidence shows that PBPs are part of multiprotein machineries whose activities are modulated by cofactors. Perturbation of these systems could lead to lethal effects. Developing screening strategies to take advantage of these mechanisms could lead to new inhibitors of PG assembly. In this paper, we present a general background on the GTs and TPs/PBPs, a survey of recent issues of bacterial resistance and a review of recent works describing new inhibitors of these enzymes.

  19. FAF and SufA: proteins of Finegoldia magna that modulate the antibacterial activity of histones.

    PubMed

    Murphy, Elizabeth C; Mohanty, Tirthankar; Frick, Inga-Maria

    2014-01-01

    Many bacterial pathogens have developed methods to overcome the defences of the host innate immune system. One such defence is the release of antimicrobial peptides (AMPs). Histones have been found to function as AMPs, in addition to their main biological function of packaging and organising DNA into nucleosomes. In this study, the Gram-positive anaerobic coccus Finegoldia magna was found to bind histones by Western blot and immunoprecipitation analysis. F. magna, which is normally a commensal of the skin and mucous membranes, is also known to act as an opportunistic pathogen and has been isolated from various clinical infection sites. It was found to bind to histones extracted from human skin epidermis through its surface and extracellular adhesion protein FAF. Through FAF binding, F. magna was protected from histone bactericidal activity. Furthermore, the histones were found to be degraded by SufA, a subtilisin-like extracellular serine protease of F. magna. Hence, the results of the present study will give more insight into how F. magna persists both as a commensal organism at the basement membrane of the skin and as an opportunistic pathogen during infection.

  20. Antibacterial properties of the sperm-binding proteins and peptides of human epididymis 2 (HE2) family; salt sensitivity, structural dependence and their interaction with outer and cytoplasmic membranes of Escherichia coli.

    PubMed Central

    Yenugu, Suresh; Hamil, Katherine G; Birse, Charles E; Ruben, Steven M; French, Frank S; Hall, Susan H

    2003-01-01

    During passage through the epididymis, sperm interact with secreted epididymal proteins that promote maturation, including the acquisition of motility and fertilization competence. Viewed previously as distinct from sperm maturation, host defence capabilities are now recognized functions of the human epididymis 2 (HE2) family of sperm-binding proteins. We analysed the potent dose and time-dependent bactericidal activity of recombinant HE2alpha, HE2beta1 and HE2beta2 and found that the full-length proteins (10 microg/ml or approximately 1 microM) caused more than a 50% decrease in Escherichia coli colony forming units within 15 min. By contrast, human beta-defensin-1, at a similar concentration, required more than 90 min to exhibit similar antibacterial activity. The epididymis-specific lipocalin, LCN6, failed to kill bacteria. Higher concentrations (25-100 microg/ml) of HE2 proteins and a longer duration of treatment resulted in near total inhibition of bacterial growth. The C-terminal peptides of HE2alpha, HEbeta1 and HEbeta2 proteins exhibited antibacterial activity similar to their full-length counterparts, indicating that the antibacterial activity of HE2 proteins resides in these C-terminal regions. Antibacterial activities of HE2 proteins and peptides were slightly inhibited by NaCl concentrations of up to 150 mM, while human beta-defensin-1 activity was nearly eliminated. Reduction and alkylation of disulphide bonds in HE2 proteins and their C-terminal peptides abolished their antibacterial activity. Consistent with the ability to kill bacteria, full-length HE2 proteins and C-terminal peptides caused rapid dose-dependent permeabilization of outer and cytoplasmic E. coli membranes. A much longer exposure time was required for human beta-defensin-1-mediated permeabilization of membranes, suggesting a possible difference in mode of action compared with the HE2 antibacterial peptides. PMID:12628001

  1. Low-frequency vibrational properties of lysozyme in sugar aqueous solutions: A Raman scattering and molecular dynamics simulation study

    NASA Astrophysics Data System (ADS)

    Lerbret, A.; Affouard, F.; Bordat, P.; Hédoux, A.; Guinet, Y.; Descamps, M.

    2009-12-01

    The low-frequency (ω <400 cm-1) vibrational properties of lysozyme in aqueous solutions of three well-known protecting sugars, namely, trehalose, maltose, and sucrose, have been investigated by means of complementary Raman scattering experiments and molecular dynamics simulations. The comparison of the Raman susceptibility χ″(ω) of lysozyme/water and lysozyme/sugar/water solutions at a concentration of 40 wt % with the χ″ of dry lysozyme suggests that the protein dynamics mostly appears in the broad peak around 60-80 cm-1 that reflects the vibrations experienced by atoms within the cage formed by their neighbors, whereas the broad shoulder around 170 cm-1 mainly stems from the intermolecular O-H⋯O stretching vibrations of water. The addition of sugars essentially induces a significant high frequency shift and intensity reduction of this band that reveal a slowing down of water dynamics and a distortion of the tetrahedral hydrogen bond network of water, respectively. Furthermore, the lysozyme vibrational densities of states (VDOS) have been determined from simulations of lysozyme in 37-60 wt % disaccharide aqueous solutions. They exhibit an additional broad peak around 290 cm-1, in line with the VDOS of globular proteins obtained in neutron scattering experiments. The influence of sugars on the computed VDOS mostly appears on the first peak as a slight high-frequency shift and intensity reduction in the low-frequency range (ω <50 cm-1), which increase with the sugar concentration and with the exposition of protein residues to the solvent. These results suggest that sugars stiffen the environment experienced by lysozyme atoms, thereby counteracting the softening of protein vibrational modes upon denaturation, observed at high temperature in the Raman susceptibility of the lysozyme/water solution and in the computed VDOS of unfolded lysozyme in water. Finally, the Raman susceptibility of sugar/water solutions and the calculated VDOS of water in the

  2. Preliminary investigations into solutal flow about growing tetragonal lysozyme crystals

    NASA Technical Reports Server (NTRS)

    Pusey, Marc; Witherow, William; Naumann, Robert

    1988-01-01

    A series of preliminary experiments were done to investigate solutal flow about growing lysozyme crystals and its effects. Density-gradient-driven flow was observed using a schlieren optical system. Crystals used ranged from 0.3 to 1.72 mm across the (110) face, and protein concentrations were from 3.7 to 23.7 mg/ml. The convective plume velocities were found to be from 10 to 50 microns/s, which correlated with those predicted to occur based upon a diffusive-convective model. When microcrystals of lysozyme, less than 20 microns across the (110) face were subjected to directed solution flows, the growth rate was found to rapidly decrease over the 8-20 h course of the experiment. Solution flow rates used ranged from 18 to 40 microns/s, and protein concentrations were from 7.3 to 11.7 mg/ml, conditions typical of larger (greater than 0.5 mm) crystals in the terminal phases of a typical crystal growth procedure.

  3. Antibacterial activity in the hemolymph of myriapods (Arthropoda).

    PubMed

    Xylander, W E; Nevermann, L

    1990-09-01

    The hemolymphs of two diplopod (Chicobolus sp. and Rhapidostreptus virgator) and two chilopod species (Lithobius forficatus and Scolopendra cingulata) were tested for the presence of antibacterial substances using Petri dish tests. The native hemolymph of all species had substances acting on living Micrococcus luteus, whereas only Rhapidostreptus, Scolopendra, and Lithobius were effective against lyophilized Micrococcus. The antibacterial activity against living Micrococcus increased after inoculation with bacteria (Enterobacter cloacae beta-12) in Chicobolus and Rhapidostreptus and also against lyophilized Micrococcus in the latter. Thus, these effects appear to be inducible. None of the myriapods tested had any bacteriostatic effect on Escherichia coli D-31 whereas the growth of gram-negative E. cloacae was inhibited. The antibacterial substances in the diplopod species were unstable when heated but were resistant to freezing. At least two antibacterial substances (a lysozyme-like one and another substance) are considered to occur in Myriapoda.

  4. Correlation of diafiltration sieving behavior of lysozyme-BSA mixtures with osmotic second virial cross-coefficients.

    PubMed

    Tessier, Peter M; Verruto, Vincent J; Sandler, Stanley I; Lenhoff, Abraham M

    2004-08-05

    The role of protein-protein interactions in membrane separations of protein mixtures remains incompletely understood, largely due to the difficulty of characterizing protein self- and, especially, cross-association. Recently, a novel technique, cross-interaction chromatography, has been developed to measure weak protein cross-association in terms of the osmotic second virial cross-coefficient. In this work the relationship between protein cross-association and the sieving behavior of lysozyme in the presence of BSA has been investigated. Sieving coefficients were measured using a stirred diafiltration cell over a range of pH and ionic strength, and a striking correlation between the lysozyme sieving and second virial cross-coefficients for BSA/lysozyme mixtures has been found: when the protein cross-interactions are most attractive (negative second virial cross-coefficient), the lysozyme sieving coefficients are lowest, and vice versa. The correlation between the sieving and second virial cross-coefficients may be due to the physically similar environments in the chromatography and filtration experiments since one protein is passed through a concentrated region of the second protein either immobilized on the column or accumulated at the membrane surface, and the migration rate of the mobile protein in both cases is influenced by protein cross-association. This study represents the first time that molecular interactions in binary mixtures have been related directly to filtration behavior, and may provide a useful approach to optimize the separation of other binary protein mixtures.

  5. Recombinant goose-type lysozyme in channel catfish: Lysozyme activity and efficacy as plasmid DNA immunostimulant against Aeromonas hydrophila infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objectives of this study were: 1) to investigate whether recombinant channel catfish lysozyme g (CC-Lys-g) produced in E. coli expression system possesses any lysozyme activity; and 2) to evaluate whether channel catfish lysozyme g plasmid DNA could be used as an immunostimulant to protect chann...

  6. Recombinant goose-type lysozyme in channel catfish: lysozyme activity and efficacy as plasmid DNA immunostimulant against Aeromonas hydrophila infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objectives of this study were: 1) to investigate whether recombinant channel catfish lysozyme g (CC-Lys-g) produced in E. coli expression system possesses any lysozyme activity; and 2) to evaluate whether channel catfish lysozyme g plasmid DNA could be used as an immunostimulant to protect chann...

  7. Investigation of β-lactam antibacterial drugs, β-lactamases, and penicillin-binding proteins with fluorescence polarization and anisotropy: a review

    NASA Astrophysics Data System (ADS)

    Shapiro, Adam B.

    2016-06-01

    This review covers the uses of fluorescence polarization and anisotropy for the investigation of bacterial penicillin binding proteins (PBPs), which are the targets of β-lactam antibacterial drugs (penicillins, cephalosporins, carbapenems, and monobactams), and of the β-lactamase enzymes that destroy these drugs and help to render bacterial pathogens resistant to them. Fluorescence polarization and anisotropy-based methods for quantitation of β-lactam drugs are also reviewed. A particular emphasis is on methods for quantitative measurement of the interactions of β-lactams and other inhibitors with PBPs and β-lactamases.

  8. “Click on the bidirectional switch”: the aptasensor for simultaneous detection of lysozyme and ATP with high sensitivity and high selectivity

    PubMed Central

    Chen, Feng; Cai, Changqun; Chen, Xiaoming; Chen, Chunyan

    2016-01-01

    A bifunctional and simple aptasensor was designed to one-spot simultaneously detect two analytes, lysozyme and ATP. The aptasensor was obtained by the electronic interaction between methyl violet (MV) and dsDNA. The dsDNA was obtained by hybridization of ATP aptamer and lysozyme aptamer. And we used the resonance light scattering (RLS) technique to detect the concentration of lysozyme and ATP. During the procedure of detection, the aptasensor works like a bidirectional switch, the corresponding side of the dsDNA will open when the target (lysozyme or ATP) “click” the aptamer, which results in corresponding RLS signal change. By the combination of the RLS technique, it is found that the changed RLS intensity was proportional to the concentration of lysozyme and ATP. The mixtures of ATP and lysozyme also met two binary function relations. The results indicated that the aptasensor could achieve simultaneous detection of ATP and lysozyme, the detection limits of ATP and lysozyme could reach 10−11 M and 10−12 M, respectively. The aptasensor shows potential application for small molecule and protein detection by RLS, it could extend the application of RLS technique. PMID:26742854

  9. Sequential separation of lysozyme, ovomucin, ovotransferrin, and ovalbumin from egg white.

    PubMed

    Abeyrathne, E D N S; Lee, H Y; Ahn, D U

    2014-04-01

    Ovalbumin, ovotransferrin, ovomucin, and lysozyme are a few of the egg white proteins that can be used as functional components. The objective of this study was to develop a simple, sequential separation method for multiple proteins from egg white. Separated proteins are targeted for human use, and thus any toxic compounds were excluded. The methods for individual components and the sequential separation were practiced in laboratory scale first, and then tested for scale-up. Lysozyme was separated first using FPC3500 cation exchange resin and then ovomucin using isoelectric precipitation. Ovalbumin and ovotransferrin were separated from the lysozyme- and ovomucin-free egg white by precipitating ovotransferrin first using 5.0% (wt/vol) (NH4)2SO4 and 2.5% (wt/vol) citric acid combination. After centrifugation, the supernatant (S1) was used for ovalbumin separation and the precipitant was dissolved in water, and reprecipitated using 2.0% ammonium sulfate (wt/vol) and 1.5% citric acid (wt/vol) combination. The precipitant was used as ovotransferrin fraction, and the supernatant (S2) was pooled with the first supernatant (S1), desalted using ultrafiltration, and then heat-treated to remove impurities. The yield of ovomucin and ovalbumen was >98% and that of ovotransferrin and lysozyme was >82% for both laboratory and scale-up preparations. The SDS-PAGE and western blotting of the separated proteins, except for ovomucin, showed >90% purity. The ELISA results indicated that the activities of separated ovalbumin, ovotransferrin, and lysozyme were >96%. The protocol separated 4 major proteins in sequence, and the method was simple and easily scaled up.

  10. An array of Escherichia coli clones over-expressing essential proteins: A new strategy of identifying cellular targets of potent antibacterial compounds

    SciTech Connect

    Xu, H. Howard . E-mail: hxu3@calstatela.edu; Real, Lilian; Bailey, Melissa Wu

    2006-11-03

    With the advancement of high throughput screening, it has become easier and faster to discover hit compounds that inhibit proliferation of bacterial cells. However, development in technologies used to identify cellular targets of potent antibacterial inhibitors has lagged behind. Here, we describe a novel strategy of target identification for antibacterial inhibitors using an array of Escherichia coli clones each over-expressing one essential protein. In a proof-of-concept study, eight essential genes were cloned into pLex5BA vector under the control of an inducible promoter. Over-expression of target proteins was confirmed. For two clones, one over-expressing FabI and the other over-expressing MurA enzymes, the host cells became 17- and 139-fold more resistant to the specific inhibitors triclosan and phosphomycin, respectively, while the susceptibility of other clones towards these inhibitors remained unchanged after induction of gene expression. Target identification via target protein over-expression was demonstrated using both mixed clone and individual clone assay formats.

  11. Osmotic second virial cross-coefficient measurements for binary combination of lysozyme, ovalbumin, and α-amylase in salt solutions.

    PubMed

    Mehta, Chirag M; White, Edward T; Litster, James D

    2013-01-01

    Interactions measurement is a valuable tool to predict equilibrium phase separation of a desired protein in the presence of unwanted macromolecules. In this study, cross-interactions were measured as the osmotic second virial cross-coefficients (B23 ) for the three binary protein systems involving lysozyme, ovalbumin, and α-amylase in salt solutions (sodium chloride and ammonium sulfate). They were correlated with solubility for the binary protein mixtures. The cross-interaction behavior at different salt concentrations was interpreted by either electrostatic or hydrophobic interaction forces. At low salt concentrations, the protein surface charge dominates cross-interaction behavior as a function of pH. With added ovalbumin, the lysozyme solubility decreased linearly at low salt concentration in sodium chloride and increased at high salt concentration in ammonium sulfate. The B23 value was found to be proportional to the slope of the lysozyme solubility against ovalbumin concentration and the correlation was explained by preferential interaction theory.

  12. Production and Ultrastructure of Lysozyme and Ethylenediaminetetraacetate-Lysozyme Spheroplasts of Escherichia coli1

    PubMed Central

    Birdsell, D. C.; Cota-Robles, E. H.

    1967-01-01

    Spheroplast production by lysozyme and ethylenediaminetetraacetate (EDTA) was examined as a means of obtaining osmotically sensitive cells for studies of enzyme localization. Physiologically young cells plasmolyzed with 0.5 m sucrose in 0.01 m tris(hydroxymethyl)aminomethane (Tris) buffer (pH 7, 8, or 9) were quantitatively converted to plasmolyzed osmotically sensitive rods after lysozyme treatment. Although such cells were osmotically sensitive, a 1:1 dilution in Tris buffer was necessary for conversion of rods into spheroplasts. Addition of EDTA resulted in a rapid conversion of the plasmolyzed spheroplasts into spherical structures devoid of a plasmolysis vacuole. These structures, which we call EDTA-lysozyme spheroplasts, contained a number of attached membranes. We believe that this conversion results from a weakening of the outer trilaminar component of the cell wall by EDTA, resulting in the collapse of the plasmolysis vacuole. Dilution of sucrose below 0.15 m also resulted in the collapse of the plasmolysis vacuole. Both the lysozyme spheroplasts and the EDTA-lysozyme spheroplasts were osmotically sensitive. Thin sections of the EDTA-lysozyme spheroplasts demonstrated that the outer trilaminar component of the cell wall was broken, exposing large areas of the cytoplasmic membrane to the environment. Images PMID:4960155

  13. Recent advances for the production and recovery methods of lysozyme.

    PubMed

    Ercan, Duygu; Demirci, Ali

    2016-12-01

    Lysozyme is an antimicrobial peptide with a high enzymatic activity and positive charges. Therefore, it has applications in food and pharmaceutical industries as an antimicrobial agent. Lysozyme is ubiquitous in both animal and plant kingdoms. Currently, egg-white lysozyme is the most commercially available form of lysozyme. The main concerns of egg-white lysozyme are high recovery cost, low activity and most importantly the immunological problems to some people. Therefore, human lysozyme production has gained importance in recent years. Scientists have developed transgenic plants, animals and microorganisms that can produce human lysozyme. Out of these, microbial production has advantages for commercial productions, because high production levels are achievable in a relatively short time. It has been reported that fermentation parameters, such as pH, temperature, aeration, are key factors to increase the effectiveness of the human lysozyme production. Moreover, purification of the lysozyme from the fermentation broth needs to be optimized for the economical production. In conclusion, this review paper covers the mechanism of lysozyme, its sources, production methods and recovery of lysozyme.

  14. Determining the Molecular Growth Mechanisms of Tetragonal Lysozyme Crystals

    NASA Technical Reports Server (NTRS)

    Li, Huayu; Nadarajah, Arunan; Konnert, John H.; Pusey, Marc L.

    1998-01-01

    Studies of the growth of tetragonal lysozyme crystals employing atomic force microscopy (AFM) have shown the advantages of this technique in investigating the growth mechanisms of protein crystals [1]. The resolution of these studies was in the micron range, which revealed surface features such as the occurrence of dislocations and 2D nucleation islands, similar to those found in inorganic systems. They clearly showed that the crystals grew by these surface growth mechanisms. However, the studies also revealed some surprising features, such as bimolecular growth step heights and pronounced growth anisotropies on the (110) face, which could not be explained. In previous studies we employed Periodic Bond Chain (PBC) theory to tetragonal lysozyme crystal growth and found that the crystals were constructed by strongly bonded molecular chains forming helices about the 43 axes [2,3]. The helices were connected to each other with weaker bonds. The growth process was shown to proceed by the formation of these 43 helices, resulting in bimolecular growth steps on the (110) face. It was also shown to explain many other observations on tetragonal lysozyme crystal growth. Although PBC analysis is not a new technique [4], it has not been widely used as the mechanisms predicted from it could not be experimentally verified. In this study the growth process of these crystals was investigated, particularly for the (110) face, employing some newly developed high resolution AFM techniques. These techniques allowed individual lysozyme molecules on the crystal faces to be resolved and predictions from PBC analyses to be tested. The analyses had shown that of the two possible packing arrangements on (110) faces, only one would actually occur. Employing the first of the newly developed techniques, these faces were scanned by high resolution AFM. The resulting images were then compared with the theoretically constructed images for the two possible packing arrangements on the (110) face

  15. A comparative study on the aggregating effects of guanidine thiocyanate, guanidine hydrochloride and urea on lysozyme aggregation

    SciTech Connect

    Emadi, Saeed Behzadi, Maliheh

    2014-08-08

    Highlights: • Lysozyme aggregated in guanidine thiocyanate (1.0 and 2.0 M). • Lysozyme aggregated in guanidine hydrochloride (4 and 5 M). • Lysozyme did not aggregated at any concentration (0.5–5 M) of urea. • Unfolding pathway is more important than unfolding per se in aggregation. - Abstract: Protein aggregation and its subsequent deposition in different tissues culminate in a diverse range of diseases collectively known as amyloidoses. Aggregation of hen or human lysozyme depends on certain conditions, namely acidic pH or the presence of additives. In the present study, the effects on the aggregation of hen egg-white lysozyme via incubation in concentrated solutions of three different chaotropic agents namely guanidine thiocyanate, guanidine hydrochloride and urea were investigated. Here we used three different methods for the detection of the aggregates, thioflavin T fluorescence, circular dichroism spectroscopy and atomic force microscopy. Our results showed that upon incubation with different concentrations (0.5, 1.0, 2.0, 3.0, 4.0, 5.0 M) of the chemical denaturants, lysozyme was aggregated at low concentrations of guanidine thiocyanate (1.0 and 2.0 M) and at high concentrations of guanidine hydrochloride (4 and 5 M), although no fibril formation was detected. In the case of urea, no aggregation was observed at any concentration.

  16. The Clostridium difficile Dlt Pathway Is Controlled by the Extracytoplasmic Function Sigma Factor σV in Response to Lysozyme

    PubMed Central

    Woods, Emily C.; Nawrocki, Kathryn L.; Suárez, Jose M.

    2016-01-01

    Clostridium difficile (also known as Peptoclostridium difficile) is a major nosocomial pathogen and a leading cause of antibiotic-associated diarrhea throughout the world. Colonization of the intestinal tract is necessary for C. difficile to cause disease. Host-produced antimicrobial proteins (AMPs), such as lysozyme, are present in the intestinal tract and can deter colonization by many bacterial pathogens, and yet C. difficile is able to survive in the colon in the presence of these AMPs. Our prior studies established that the Dlt pathway, which increases the surface charge of the bacterium by addition of d-alanine to teichoic acids, is important for C. difficile resistance to a variety of AMPs. We sought to determine what genetic mechanisms regulate expression of the Dlt pathway. In this study, we show that a dlt null mutant is severely attenuated for growth in lysozyme and that expression of the dltDABC operon is induced in response to lysozyme. Moreover, we found that a mutant lacking the extracytoplasmic function (ECF) sigma factor σV does not induce dlt expression in response to lysozyme, indicating that σV is required for regulation of lysozyme-dependent d-alanylation of the cell wall. Using reporter gene fusions and 5′ RACE (rapid amplification of cDNA ends) analysis, we identified promoter elements necessary for lysozyme-dependent and lysozyme-independent dlt expression. In addition, we observed that both a sigV mutant and a dlt mutant are more virulent in a hamster model of infection. These findings demonstrate that cell wall d-alanylation in C. difficile is induced by lysozyme in a σV-dependent manner and that this pathway impacts virulence in vivo. PMID:27068095

  17. Amoebicidal Activity of Milk, Apo-lactoferrin, sIgA and Lysozyme

    PubMed Central

    León-Sicairos, Nidia; López-Soto, Fernando; Reyes-López, Magda; Godínez-Vargas, Delfino; Ordaz-Pichardo, Cynthia; de la Garza, Mireya

    2006-01-01

    Objectives: To identify amoebicidal components in human milk and the effect of iron on the amoebicidal activity. Design: Investigation in axenic cultures of Entamoeba histolytica trophozoites. Methods: Amoebas were treated with 5%–20% of human, bovine and swine milk, with 10% of human milk fractions (i.e., casein, proteins except casein and fat) or with 1 mg/ml of human milk apo-lactoferrin, human secretory immunoglobulin type A (sIgA) and chicken egg-white lysozyme (i.e., purified proteins). Milk proteins were detected using immunoblot. Confocal microscopy was used to define the interaction of milk proteins (100 μM each) and amoebas. Experiments were done at least three times in triplicate, and mean and standard deviations were calculated. Results: Human and bovine milk were amoebicidal showing a concentration-dependent effect. The amoebicidal effect was increased in the absence of iron. Milk protein fractions, with the exception of casein, were the components responsible for the amoebicidal activity found. Apo-lactoferrin, sIgA and lysozyme were identified in the amoebicidal milk protein fraction. Apo-lactoferrin showed the major amoebicidal effect. These proteins, either alone or in combination, showed a killing effect on the trophozoites. They bound to the amoebic membrane causing cell rounding, lipid disruption and damage. Conclusions: Milk proteins such as apo-lactoferrin, sIgA and lysozyme are able to kill Entamoeba histolytica trophozoites. This study confirms the importance of feeding breast milk to newborns. PMID:16809402

  18. Spherulitic growth of hen egg-white lysozyme crystals.

    PubMed

    Heijna, Maurits C R; Theelen, Mirjam J; van Enckevort, Willem J P; Vlieg, Elias

    2007-02-22

    In protein crystallography, spherulites are considered the result of a failed crystallization experiment. Understanding the formation of these structures may contribute to finding methods to prevent their formation. Here, we present an in situ study on lysozyme spherulites grown from sodium nitrate and sodium thiocyanate solutions, investigating their morphology and growth kinetics using optical microscopy. In a morphodrom, we indicate the conditions at which spherulites form for the lysozyme-nitrate system, showing that liquid-liquid phase separation is not a prerequisite to form sheaflike spherulites and that supersaturation is not the only factor determining their creation. Despite their sheaflike morphology, the spherulites all appear to be formed through heterogeneous nucleation. The spherulites are of a new polymorphic form and are less stable than the monoclinic form. For a single needle, growth kinetics indicate surface processes to be the rate-limiting step during growth, but for an entire spherulite volume, diffusion still plays a role. Spherulites simulated by using a time-dependent, tip-splitting model are found to compare well to experimentally observed spherulites.

  19. Solvent-induced lysozyme gels: rheology, fractal analysis, and sol-gel kinetics.

    PubMed

    da Silva, Marcelo A; Arêas, Elizabeth P G

    2005-09-15

    In this work, the gelation kinetics and fractal character of lysozyme gel matrices developed in tetramethylurea (TMU)-water media were investigated. Gelation times were determined from the temporal crossover point between the storage, G', and loss, G'', moduli, as a function of the binary solvent composition and of protein concentration. The inverse dependence of the upper limit of the linear viscoelastic region (gamma0) on protein concentration indicate that the lysozyme gels belong to the "strong link" kind, a gel category where interparticle links are stronger than intraparticle ones. Lysozyme gel fractal dimensions (Df) were determined from the analysis of rheological data according to a scaling theory by Shih et al. [Phys. Rev. A 42 (1990) 4772-4779] and were found to be compatible with a diffusion-limited cluster-aggregation kinetics (DLCA) for lysozyme gels formed at the TMU mass fraction in the binary organic-aqueous solvent, wTMU=0.9, and with a reaction-limited cluster aggregation kinetics (RLCA) for wTMU in the 0.6< or =wTMU< or =0.8 range.

  20. Stabilization of lysozyme by benzyl alcohol: surface tension and thermodynamic parameters.

    PubMed

    Goyal, Monu Kumari; Roy, Ipsita; Amin, Aeshna; Banerjee, Uttam Chand; Bansal, Arvind Kumar

    2010-10-01

    The aim of the study was to understand the effect of benzyl alcohol on biological activity, aggregation behavior, denaturant and heat-induced unfolding of lysozyme. Compatibility studies of lysozyme carried out with a number of anti-microbial preservatives, indicated benzyl alcohol to be the best suppressor of protein aggregation against heat stress. The effect of this preservative was checked at various pH values ranging from 4.0 to 9.0. In spite of reducing the thermal denaturation temperature (T(m)) at all pH values, benzyl alcohol had a stabilizing effect on lysozyme in terms of retaining the biological activity when the enzyme was incubated at 75 degrees C. The reduction in T(m) with increasing benzyl alcohol concentration was correlated with decreasing surface tension of surrounding medium. A detailed thermodynamic study of lysozyme in the presence of benzyl alcohol was carried out at pH 6.2. Change in Gibb's free energy of thermal unfolding at 25 degrees C was found to remain constant in the presence of benzyl alcohol, indicating no interaction of benzyl alcohol with the native protein at room temperature. Both the enthalpy and entropy change at mid point of thermal unfolding were found to increase in the presence of benzyl alcohol indicating the stabilization of partially unfolded state.

  1. Characterization of heat induced spherulites of lysozyme reveals new insight on amyloid initiation

    PubMed Central

    Sharma, Pankaj; Verma, Neha; Singh, Pradip Kumar; Korpole, Suresh; Ashish

    2016-01-01

    Here, we report results obtained during our experiments to visualize how heat transforms globular protein, lysozyme into building block of β-amyloids. Light scattering experiments showed formation of lower order associated species around 50–70 °C followed by rapid cooperativity to β-amyloid fibrils. Interestingly, crystallization drops set at higher temperatures either led to aggregates or spherulites. The latter possess an amorphous β-fibril rich core with thin crystalline needles projecting outwards. Diffraction of the crystalline outgrowths revealed novel dimers and trimers of lysozyme where individual chains were similar to monomer with marginal gain in β-sheet content. Importantly, analysis of Amide I stretching frequencies showed that protein loses its secondary structure at temperatures higher than where we obtained crystals followed by rapid gain in β-sheet content. Interestingly, attempts to use the needles as seeds for more crystals led to “broom-like” fibril formations at the ends. Further, aggregation inhibitors like arginine and benzyl alcohol completely obliterated spherulites formation during crystallization. Refinement of crystals of lysozyme in presence of these molecules showed these small molecules bind to the interfaces of heat associated dimers and trimers. Overall our work concludes that heat induced weakly associated structures of lysozyme are the first step towards its amyloid formation. PMID:26926993

  2. The effect of lysozyme amyloid fibrils on cytochrome c-lipid interactions.

    PubMed

    Gorbenko, Galyna; Trusova, Valeriya; Sood, Rohit; Molotkovsky, Julian; Kinnunen, Paavo

    2012-10-01

    Protein polymerization into ordered fibrillar structures (amyloid fibrils) is currently associated with a range of pathological conditions. Recent studies clearly indicate that amyloid cytotoxicity is provoked by a continuum of cross-β-sheet aggregates including mature fibrils. In view of the possible diversity of cytotoxicity mechanisms, the present study addressed the question of whether protein conversion into amyloid fibrils can modify its competitive membrane adsorption behavior. Using a combination of resonance energy transfer, dynamic light scattering and fluorescence quenching techniques, the competitive binding of either monomeric or polymerized lysozyme, and cytochrome c to the model lipid membranes composed of phosphatidylcholine mixtures with varying proportions of phosphatidylglycerol, phosphatidylserine or cardiolipin has been studied. The ability of fibrillar lysozyme to induce dissociation of cytochrome c from the membrane binding sites proved to be markedly stronger than that of its monomeric counterpart, with desorption process displaying cooperativity features upon increasing the charge of lipid bilayer. The decreased efficiency of tryptophan fluorescence quenching by acrylamide and short-wavelength shift of emission maximum observed upon membrane binding of lysozyme fibrils were rationalized in terms of fluorophore transfer into interfacial bilayer region. It is hypothesized that electrostatic interactions play predominant role in determining the lipid-associating and competitive abilities of fibrillar lysozyme.

  3. Study of lysozyme mobility and binding free energy during adsorption on a graphene surface

    SciTech Connect

    Nakano, C. Masato; Ma, Heng; Wei, Tao

    2015-04-13

    Understanding protein adsorption is a key to the development of biosensors and anti-biofouling materials. Hydration essentially controls the adsorption process on hydrophobic surfaces, but its effect is complicated by various factors. Here, we present an ideal model system to isolate hydration effects—lysozyme adsorption on a flat hydrophobic graphene surface. Our all-atom molecular dynamics and molecular-mechanics/Poisson-Boltzmann surface area computation study reveal that lysozyme on graphene displays much larger diffusivity than in bulk water. Protein's hydration free energy within the first hydration shell is dominated by the protein-water electrostatic interactions and acts as an energy barrier for protein adsorption. On the other hand, the surface tension, especially that from the hydrophobic graphene, can effectively weaken the barrier to promote adsorption.

  4. Lysozyme- and chitinase activity in latex bearing plants of genus Euphorbia--A contribution to plant defense mechanism.

    PubMed

    Sytwala, Sonja; Günther, Florian; Melzig, Matthias F

    2015-10-01

    Occurrence of latices in plants is widespread, there are 40 families of plants characterized to establish lactiferous structures. Latices exhibit a constitutive part of plant defense due to the stickiness. The appearance of proteins incorporated in latices is well characterized, and hydrolytic active proteins are considerable. A lot of plants constitute so-called pathogenesis-related (PR) proteins, to overcome stressful conditions. In our investigation we are focused on latex bearing plants of Euphorbiaceae Juss., and investigated the appearance of chitinase- and lysozyme activity in particular. The present outcomes represent a comprehensive study, relating to the occurrence of lysozyme and chitinase activity of genus Euphorbia at the first time. 110 different species of genus Euphorbia L. were tested, and the appearance of chitinase and lysozyme were determined in different quantities. The appearance itself, and the physicochemical properties of latices indicate an efficient interaction for plant defense against pathogen attack.

  5. Crystallization of Chicken Egg White Lysozyme from Assorted Sulfate Salts

    NASA Technical Reports Server (NTRS)

    Forsythe, Elizabeth L.; Snell, Edward H.; Malone, Christine C.; Pusey, Marc L.

    1999-01-01

    Chicken egg white lysozyme has been found to crystallize from ammonium, sodium, potassium, rubidium, magnesium, and manganese sulfates at acidic and basic pH, with protein concentrations from 60 to 190 mg/ml. Crystals have also been grown at 4 C in the absence of any other added salts using isoionic lysozyme which was titrated to pH 4.6 with dilute sulfuric acid. Four different crystal forms have been obtained, depending upon the temperature, protein concentration, and precipitating salt employed. Crystals grown at 15 C were generally tetragonal, with space group P4(sub 3)2(sub 1)2. Crystallization at 20 C typically resulted in the formation of orthorhombic crystals, space group P2(sub 1)2(sub 1)2(sub 1). The tetragonal reversible reaction orthorhombic transition appeared to be a function of both the temperature and protein concentration, occurring between 15 and 20 C and between 100 and 125 mg/ml protein concentration. Crystallization from 1.2 M magnesium sulfate at pH 7.8 gave a trigonal crystal, space group P3(sub 1)2(sub 1), a = b = 87.4, c = 73.7, gamma = 120 deg, which diffracted to 2.8 A. Crystallization from ammonium sulfate at pH 4.6, generally at lower temperatures, was also found to result in a monoclinic form. space group C2, a = 65.6, b = 95.0, c = 41.2, beta = 119.2 deg. A crystal of approximately 0.2 x 0.2 x 0.5 mm grown from bulk solution diffracted to approximately 3.5 A.

  6. Hydration dependence of conformational dielectric relaxation of lysozyme.

    PubMed

    Knab, Joseph; Chen, Jing-Yin; Markelz, Andrea

    2006-04-01

    Dielectric response of hen egg white lysozyme is measured in the far infrared (5-65 cm-1, 0.15-1.95 THz, 0.6-8.1 meV) as a function of hydration. The frequency range is associated with collective vibrational modes of protein tertiary structure. The observed frequency dependence of the absorbance is broad and glass-like. For the entire frequency range, there is a slight increase in both the absorbance and index of refraction with increasing hydration for <0.27 h (mass of H2O per unit mass protein). At 0.27 h, the absorbance and index begin to increase more rapidly. This transition corresponds to the point where the first hydration shell is filled. The abrupt increase in dielectric response cannot be fully accounted for by the additional contribution to the dielectric response due to bulk water, suggesting that the protein has not yet achieved its fully hydrated state. The broad, glass-like response suggests that at low hydrations, the low frequency conformational hen egg white lysozyme dynamics can be described by a dielectric relaxation model where the protein relaxes to different local minima in the conformational energy landscape. However, the low frequency complex permittivity does not allow for a pure relaxational mechanism. The data can best be modeled with a single low frequency resonance (nu approximately 120 GHz=4 cm-1) and a single Debye relaxation process (tau approximately .03-.04 ps). Terahertz dielectric response is currently being considered as a possible biosensing technique and the results demonstrate the required hydration control necessary for reliable biosensor applications.

  7. Investigation of effects of terpene skin penetration enhancers on stability and biological activity of lysozyme.

    PubMed

    Varman, Rahul M; Singh, Somnath

    2012-12-01

    The transport of proteins through skin can be facilitated potentially by using terpenes as chemical enhancers. However, we do not know about the effects of these enhancers on the stability and biological activity of proteins which is crucial for the development of safe and efficient formulations. Therefore, this project investigated the effects of terpene-based skin penetration enhancers which are reported as nontoxic to the skin (e.g., limonene, p-cymene, geraniol, farnesol, eugenol, menthol, terpineol, carveol, carvone, fenchone, and verbenone), on the conformational stability and biological activity of a model protein lysozyme. Terpene (5% v/v) was added to lysozyme solution and kept for 24 h (the time normally a transdermal patch remains) for investigating conformational stability profiles and biological activity. Fourier transform infrared spectrophotometer was used to analyze different secondary structures, e.g., α-helix, β-sheet, β-turn, and random coil. Conformational changes were also monitored by differential scanning calorimeter by determining midpoint transition temperature (Tm) and calorimetric enthalpy (ΔH). Biological activity of lysozyme was determined by measuring decrease in A (450) when it was added to a suspension of Micrococcus lysodeikticus. The results of this study indicate that terpenes 9, 10, and 11 (carvone, L-fenchone, and L-verbenone) decreased conformational stability and biological activity of lysozyme significantly (p < 0.05) less than other terpenes used in this study. It is concluded that smaller terpenes containing ketones with low lipophilicity (log K (ow) ∼2.00) would be optimal for preserving conformational stability and biological activity of lysozyme in a transdermal formulation containing terpene as permeation enhancer.

  8. The Invertebrate Lysozyme Effector ILYS-3 Is Systemically Activated in Response to Danger Signals and Confers Antimicrobial Protection in C. elegans

    PubMed Central

    Gravato-Nobre, Maria João; Vaz, Filipa; Filipe, Sergio; Chalmers, Ronald; Hodgkin, Jonathan

    2016-01-01

    Little is known about the relative contributions and importance of antibacterial effectors in the nematode C. elegans, despite extensive work on the innate immune responses in this organism. We report an investigation of the expression, function and regulation of the six ilys (invertebrate-type lysozyme) genes of C. elegans. These genes exhibited a surprising variety of tissue-specific expression patterns and responses to starvation or bacterial infection. The most strongly expressed, ilys-3, was investigated in detail. ILYS-3 protein was expressed constitutively in the pharynx and coelomocytes, and dynamically in the intestine. Analysis of mutants showed that ILYS-3 was required for pharyngeal grinding (disruption of bacterial cells) during normal growth and consequently it contributes to longevity, as well as being protective against bacterial pathogens. Both starvation and challenge with Gram-positive pathogens resulted in ERK-MAPK-dependent up-regulation of ilys-3 in the intestine. The intestinal induction by pathogens, but not starvation, was found to be dependent on MPK-1 activity in the pharynx rather than in the intestine, demonstrating unexpected communication between these two tissues. The coelomocyte expression appeared to contribute little to normal growth or immunity. Recombinant ILYS-3 protein was found to exhibit appropriate lytic activity against Gram-positive cell wall material. PMID:27525822

  9. Kinetics of supersaturation decay in the crystallization of lysozyme

    NASA Astrophysics Data System (ADS)

    Kim, Y. W.; Barlow, D. A.; Caraballo, K. G.; Baird, J. K.

    The molecular architecture of proteins can be determined by analysing the X-ray diffraction patterns of their crystals. The technology of X-ray crystallography has reached the point, however, where the determination of the structure of a given crystal is controlled by the limited availability of the crystals themselves. Proteins can often be crystallized from pH buffered aqueous solutions of strong electrolytes. When dissolved protein in solution is more stable than crystalline protein, the appearance of crystals can be said to be under thermodynamic control. If, on the other hand, the crystals are more stable than the dissolved protein, and still crystals are slow to appear, the crystallization can be said to be under kinetic control. Using dilatometry, we have followed the rate of decay of the protein supersaturation in crystallizing solutions of chicken egg-white lysozyme under conditions of kinetic control. We have found that the rate of decay of the supersaturation is first order in the supersaturation and that the rate constant is independent of the initial protein concentration, but increases with increasing pH, decreasing temperature, and with increasing concentrations of sodium chloride and buffer salt. We correlate these observed trends in the rate constant with related trends in the solubility and surface charge density of the crystals. We conclude that the rate constant for supersaturation decay is inversely proportional to the protein solubility.

  10. A gene (tmpA) for an efflux protein of the transporter family III from Brevibacterium linens OC2, an antibacterial substance-producing strain.

    PubMed

    Boucabeille, C; Simonet, J M; Henckes, G

    1999-06-01

    A gene (tmpA) encoding a putative transmembrane protein has been cloned from B. linens OC2, an antibacterial substance-producing strain. The deduced TmpA protein sequence shares similarities to members of the transporter family III exploiting the transmembrane proton gradient to provide export of toxic compounds such as antiseptics or antibiotics. Northern blot analysis indicated that tmpA gene is expressed. Length of RNA messenger and overlapping of ORFs upstream tmpA gene suggested that it might belong to an operon. The tmpA gene is unusual among B. linens species since it was not detected among eight B. linens collection strains and 40 B. linens industrial strains.

  11. Antibacterial products of marine organisms.

    PubMed

    Ng, Tzi Bun; Cheung, Randy Chi Fai; Wong, Jack Ho; Bekhit, Adnan A; Bekhit, Alaa El-Din

    2015-05-01

    Marine organisms comprising microbes, plants, invertebrates, and vertebrates elaborate an impressive array of structurally diverse antimicrobial products ranging from small cyclic compounds to macromolecules such as proteins. Some of these biomolecules originate directly from marine animals while others arise from microbes associated with the animals. It is noteworthy that some of the biomolecules referred to above are structurally unique while others belong to known classes of compounds, peptides, and proteins. Some of the antibacterial agents are more active against Gram-positive bacteria while others have higher effectiveness on Gram-negative bacteria. Some are efficacious against both Gram-positive and Gram-negative bacteria and against drug-resistant strains as well. The mechanism of antibacterial action of a large number of the chemically identified antibacterial agents, possible synergism with currently used antibiotics, and the issue of possible toxicity on mammalian cells and tissues await elucidation. The structural characteristics pivotal to antibacterial activity have been ascertained in only a few studies. Demonstration of efficacy of the antibacterial agents in animal models of bacterial infection is highly desirable. Structural characterization of the active principles present in aqueous and organic extracts of marine organisms with reportedly antibacterial activity would be desirable.

  12. Tracing lysozyme-lipid interactions with long-wavelength squaraine dyes.

    PubMed

    Ioffe, Valeriya M; Gorbenko, Galyna P; Kinnunen, P K J; Tatarets, Anatoliy L; Kolosova, Olga S; Patsenker, Leonid D; Terpetschnig, Ewald A

    2007-01-01

    The applicability of the two newly commercial available squaraine labels Square-670-NHS and Seta-635-NHS to exploring protein-lipid interactions has been evaluated. The labels were conjugated to lysozyme (Lz) (squaraine-lysozyme conjugates below referred to as Square-670-Lz and Seta-635-Lz), a structurally well-characterized small globular protein displaying the ability to interact both, electrostatically and hydrophobically with lipids. The lipid component of the model systems was represented by lipid vesicles composed of zwitterionic lipids egg yolk phosphatidylcholine (PC) and 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and their mixtures with anionic lipids either beef heart cardiolipin (CL) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), respectively. Fluorescence intensity of Square-670-Lz was found to decrease upon association with lipid bilayer, while the fluorescence intensity of Seta-635-Lz displayed more complex behavior depending on lipid-to-protein molar ratio. Covalent coupling of squaraine labels to lysozyme exerts different influence on the properties of dye-protein conjugate. It was suggested that Square-670-NHS covalent attachment to Lz molecule enhances protein propensity for self-association, while squaraine label Seta-635-NHS is sensitive to different modes of lysozyme-lipid interactions-within the L:P range 6-11, when hydrophobic protein-lipid interactions are predominant, an aggregation of membrane-bound protein molecules takes place, thereby decreasing the fluorescence intensity of Seta-635-Lz. At higher L:P values (from 22 to 148) when electrostatic interactions are enhanced fluorescence intensity of Seta-635-Lz increases with increasing lipid concentrations.

  13. Growth and dissolution kinetics of tetragonal lysozyme

    NASA Technical Reports Server (NTRS)

    Monaco, L. A.; Rosenberger, F.

    1993-01-01

    The growth and dissolution kinetics of lysozyme in a 25 ml solution bridge inside a closed growth cell was investigated. It was found that, under all growth conditions, the growth habit forming (110) and (101) faces grew through layer spreading with different growth rate dependence on supersaturation/temperature. On the other hand, (100) faces which formed only at low temperatures underwent a thermal roughening transition around 12 C.

  14. Isolation of hen egg white lysozyme, ovotransferrin and ovalbumin, using a quaternary ammonium bound to a highly crosslinked agarose matrix.

    PubMed

    Vachier, M C; Piot, M; Awadé, A C

    1995-02-03

    A single-step anion-exchange chromatographic separation of egg white proteins was carried out using a Q Sepharose Fast Flow column. The separation resulted in the isolation of two lysozyme peaks with purities of ca. 99 and 88%, one peak of ovotransferrin purified to ca. 75% and two ovalbumin peaks with purities of ca. 54 and 98%. Recoveries were estimated to be ca. 60, 100 and 83% for lysozyme, ovotransferrin and ovalbumin, respectively. The amino acid compositions of all collected peaks have also been determined. This confirmed the identity of some of the proteins contained in these peaks.

  15. Crystallization of lysozyme with (R)-, (S)- and (RS)-2-methyl-2,4-pentanediol

    PubMed Central

    Stauber, Mark; Jakoncic, Jean; Berger, Jacob; Karp, Jerome M.; Axelbaum, Ariel; Sastow, Dahniel; Buldyrev, Sergey V.; Hrnjez, Bruce J.; Asherie, Neer

    2015-01-01

    Chiral control of crystallization has ample precedent in the small-molecule world, but relatively little is known about the role of chirality in protein crystallization. In this study, lysozyme was crystallized in the presence of the chiral additive 2-methyl-2,4-pentanediol (MPD) separately using the R and S enantiomers as well as with a racemic RS mixture. Crystals grown with (R)-MPD had the most order and produced the highest resolution protein structures. This result is consistent with the observation that in the crystals grown with (R)-MPD and (RS)-MPD the crystal contacts are made by (R)-MPD, demonstrating that there is preferential interaction between lysozyme and this enantiomer. These findings suggest that chiral interactions are important in protein crystallization. PMID:25760593

  16. Production, crystallization and X-ray characterization of chemically glycosylated hen egg-white lysozyme

    SciTech Connect

    López-Jaramillo, F. J.; Pérez-Banderas, F.; Hernández-Mateo, F.; Santoyo-González, F.

    2005-04-01

    The feasibility of glycosylation post-purification has been demonstrated by introducing glucose into the model protein lysozyme via a novel reaction that is compatible with biological samples. The crystallization of glycoproteins is one of the challenges to be confronted by the crystallographic community in the frame of what is known as glycobiology. The state of the art for the crystallization of glycoproteins is not promising and removal of the carbohydrate chains is generally suggested since they are flexible and a source of heterogeneity. In this paper, the feasibility of introducing glucose into the model protein hen egg-white lysozyme via a post-purification glycosylation reaction that may turn any protein into a model glycoprotein whose carbohydrate fraction can be manipulated is demonstrated.

  17. The Non-Core Regions of Human Lysozyme Amyloid Fibrils Influence Cytotoxicity

    PubMed Central

    Mossuto, Maria F.; Dhulesia, Anne; Devlin, Glyn; Frare, Erica; Kumita, Janet R.; de Laureto, Patrizia Polverino; Dumoulin, Mireille; Fontana, Angelo; Dobson, Christopher M.; Salvatella, Xavier

    2010-01-01

    Identifying the cause of the cytotoxicity of species populated during amyloid formation is crucial to understand the molecular basis of protein deposition diseases. We have examined different types of aggregates formed by lysozyme, a protein found as fibrillar deposits in patients with familial systemic amyloidosis, by infrared spectroscopy, transmission electron microscopy, and depolymerization experiments, and analyzed how they affect cell viability. We have characterized two types of human lysozyme amyloid structures formed in vitro that differ in morphology, molecular structure, stability, and size of the cross-β core. Of particular interest is that the fibrils with a smaller core generate a significant cytotoxic effect. These findings indicate that protein aggregation can give rise to species with different degree of cytotoxicity due to intrinsic differences in their physicochemical properties. PMID:20624399

  18. Large-scale production of functional human lysozyme from marker-free transgenic cloned cows.

    PubMed

    Lu, Dan; Liu, Shen; Ding, Fangrong; Wang, Haiping; Li, Jing; Li, Ling; Dai, Yunping; Li, Ning

    2016-03-10

    Human lysozyme is an important natural non-specific immune protein that is highly expressed in breast milk and participates in the immune response of infants against bacterial and viral infections. Considering the medicinal value and market demand for human lysozyme, an animal model for large-scale production of recombinant human lysozyme (rhLZ) is needed. In this study, we generated transgenic cloned cows with the marker-free vector pBAC-hLF-hLZ, which was shown to efficiently express rhLZ in cow milk. Seven transgenic cloned cows, identified by polymerase chain reaction, Southern blot, and western blot analyses, produced rhLZ in milk at concentrations of up to 3149.19 ± 24.80 mg/L. The purified rhLZ had a similar molecular weight and enzymatic activity as wild-type human lysozyme possessed the same C-terminal and N-terminal amino acid sequences. The preliminary results from the milk yield and milk compositions from a naturally lactating transgenic cloned cow 0906 were also tested. These results provide a solid foundation for the large-scale production of rhLZ in the future.

  19. Behavior of lysozyme adsorbed onto biological liquid crystal lipid monolayer at the air/water interface

    NASA Astrophysics Data System (ADS)

    Lu, Xiaolong; Shi, Ruixin; Hao, Changchun; Chen, Huan; Zhang, Lei; Li, Junhua; Xu, Guoqing; Sun, Runguang

    2016-09-01

    The interaction between proteins and lipids is one of the basic problems of modern biochemistry and biophysics. The purpose of this study is to compare the penetration degree of lysozyme into 1,2-diapalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethano-lamine (DPPE) by analyzing the data of surface pressure-area (π-A) isotherms and surface pressure-time (π-T) curves. Lysozyme can penetrate into both DPPC and DPPE monolayers because of the increase of surface pressure at an initial pressure of 15 mN/m. However, the changes of DPPE are larger than DPPC, indicating stronger interaction of lysozyme with DPPE than DPPC. The reason may be due to the different head groups and phase state of DPPC and DPPE monolayers at the surface pressure of 15 mN/m. Atomic force microscopy reveals that lysozyme was absorbed by DPPC and DPPE monolayers, which leads to self-aggregation and self-assembly, forming irregular multimers and conical multimeric. Through analysis, we think that the process of polymer formation is similar to the aggregation mechanism of amyloid fibers. Project supported by the National Natural Science Foundation of China (Grant Nos. 21402114 and 11544009), the Natural Science Basic Research Plan in Shaanxi Province of China (Grant No. 2016JM2010), the Fundamental Research Funds for the Central Universities of China (Grant No. GK201603026), and the National University Science and Technology Innovation Project of China (Grant No. 201610718013).

  20. Thermodynamic studies of partitioning behavior of lysozyme and conalbumin in aqueous two-phase systems.

    PubMed

    de Sousa, Rita de Cássia Superbi; Coimbra, Jane Sélia dos Reis; da Silva, Luis Henrique Mendes; da Silva, Maria do Carmo Hespanhol; Rojas, Edwin Elard Garcia; Vicente, Antonio António Augusto

    2009-08-15

    The objective of this study was to determine the thermodynamic parameters (Delta(tr)G, Delta(tr)H and Delta(tr)S) associated with lysozyme and conalbumin partitioning in aqueous two-phases systems (ATPS). Influence of salt type and polyethylene glycol (PEG) concentrations on the partition coefficient of lysozyme and conalbumin from egg white was studied. The evaluated ATPS were composed of PEG 1500 and inorganic salts (sodium citrate and sodium sulfate) at a temperature of 25 degrees C and pH 7.0, with PEG 1500 g mol(-1) concentrations of 14%, 16% and 18% (mass basis). Partitioning of lysozyme in PEG-citrate ATPS was enthalpically driven, however the PEG-sulfate ATPS was entropically driven. The tested systems can be employed for the separation of these two proteins in egg white, due to the fact that lysozyme migrates toward the polymeric phase and conalbumin to the saline phase in both ATPS. A high recovery of conalbumin in the saline phase of the PEG-sulfate ATPS was determined to be enthalpically driven.

  1. Low-frequency spectroscopic analysis of monomeric and fibrillar lysozyme.

    PubMed

    Zakaria, Hidayatul A; Fischer, Bernd M; Bradley, Andrew P; Jones, Inke; Abbott, Derek; Middelberg, Anton P J; Falconer, Robert J

    2011-03-01

    Terahertz time-domain spectroscopy (THz-TDS) and Fourier transform infrared (FT-IR) spectroscopy were used to generate far-infrared and low-frequency spectral measurements of monomeric lysozyme and lysozyme fibrils. The formation of lysozyme fibrils was verified by the Thioflavin T assay and transmission electron microscopy (TEM). It was evident in the FT-IR spectra that between 150 and 350 cm(-1) the two spectra diverge, with the lysozyme fibrils showing higher absorbance intensity than the monomeric form. The broad absorption phenomenon is likely due to light scattered from the fibrillar architecture of lysozyme fibrils as supported by simulation of Rayleigh light scattering. The lack of discrete phonon-like peaks suggest that far-infrared spectroscopy cannot detect vibrational modes between the highly ordered hydrogen-bonded beta-pleated sheets of the lysozyme subunit.

  2. The Hcp proteins fused with diverse extended-toxin domains represent a novel pattern of antibacterial effectors in type VI secretion systems.

    PubMed

    Ma, Jiale; Pan, Zihao; Huang, Jinhu; Sun, Min; Lu, Chengping; Yao, Huochun

    2017-01-06

    The type VI secretion system (T6SS) is a widespread molecular weapon deployed by many bacterial species to target eukaryotic host cells or rival bacteria. Using a dynamic injection mechanism, diverse effectors can be delivered by T6SS directly into recipient cells. Here, we report a new family of T6SS effectors encoded by extended Hcps carrying diverse toxin domains. Bioinformatic analyses revealed that these Hcps with C-terminal extension toxins, designated as Hcp-ET, exist widely in the Enterobacteriaceae. To verify our findings, Hcp-ET1 was tested for its antibacterial effect, and showed effective inhibition of target cell growth via the predicted HNH-DNase activity by T6SS-dependent delivery. Further studies showed that Hcp-ET2 mediated interbacterial antagonism via a Tle1 phospholipase (encoded by DUF2235 domain) activity. Notably, comprehensive analyses of protein homology and genomic neighborhoods revealed that Hcp-ET3-4 is fused with 2 toxin domains (Pyocin S3 and Colicin-DNase) C-terminally, and its encoding gene is followed 3 duplications of the cognate immunity genes. However, some bacteria encode a separated hcp-et3 and an orphan et4 (et4O1) genes caused by a termination-codon mutation in the fusion region between Pyocin S3 and Colicin-DNase encoding fragments. Our results demonstrated that both of these toxins had antibacterial effects. Further, all duplications of the cognate immunity protein contributed to neutralize the DNase toxicity of Pyocin S3 and Colicin, which has not been reported previously. In conclusion, we propose that Hcp-ET proteins are polymorphic T6SS effectors, and thus present a novel encoding pattern of T6SS effectors.

  3. Crystal Growth of Hen Egg-White Lysozyme (HEWL) under Various Gravity Conditions

    NASA Astrophysics Data System (ADS)

    Pan, Weichun; Xu, Jin; Tsukamoto, Katsuo; Koizumi, Masako; Yamazaki, Tomoya; Zhou, Ru; Li, Ang; Fu, Yuying

    2013-08-01

    Motivated by the enhancement of protein quality under microgravity condition, the behaviors of crystal growth under various gravity conditions have been monitored via Foton Satellite and parabolic flight. We found that the normal growth rate and the step velocity would be enhanced only at high protein concentration. Although the difference of diffusion between monomer lysozyme molecule and main impurity species in HWEL dimer may be able to explain this enhancement in long period at high protein concentration, it is not valid at low lysozyme concentration and it can't explain the results obtained by parabolic flight, in which microgravity condition maintained only about 20 s. In order to compromise this contradiction, cluster, universal existing in protein solution, has been picked up. The dynamic light scattering technique figured out dimer is served as the seed for cluster formation. Due to its large size, cluster keeps still under microgravity. Via this mechanism, the purification of lysozyme above crystal surface has been achieved. We also found the two supergravity (˜1.5 g) periods immediately before and after microgravity period have different effects on the step velocity. The pre-MG period depresses the step velocity while the post-MG enhances it. This odd phenomenon ascribes to two factors: (1) the flow rate modification and (2) the purity of protein solution immediate above crystal surface.

  4. Introduction of Ca(2+)-binding amino-acid sequence into the T4 lysozyme.

    PubMed

    Leontiev, V V; Uversky, V N; Permyakov, E A; Murzin, A G

    1993-03-05

    The 51-62 loop of T4 phage lysozyme was altered by site-directed mutagenesis to obtain maximal homology with the typical EF-hand motif. A Ca(2+)-binding site was designed and created by replacing both Gly-51 and Asn-53 with aspartic acid. The mutant T4 lysozyme (G51D/N53D) was expressed in Escherichia coli. The activity of the G51D/N53D-mutant was about 60% of that of the wild-type protein. This mutant can bind Ca2+ ions specifically, while the effective dissociation constant was essentially greater than that of the EF-hand proteins. Stability of the G51D/N53D-mutant apo-form to urea- or temperature-induced denaturation was the same as that of the wild-type protein. In the presence of Ca2+ ions in solution the stability of the mutant T4 phage lysozyme was less than that of the wild-type protein. It is suggested that the binding of Ca2+ by the mutant is accompanied by the considerable conformational changes in the 'corrected' loop, which can lead to the Ca(2+)-induced destabilization of the protein.

  5. The effects of temperature and NaCl concentration on tetragonal lysozyme face growth rates

    NASA Technical Reports Server (NTRS)

    Forsythe, Elizabeth; Pusey, Marc Lee

    1994-01-01

    Measurements were made of the (110) and (101) face growth rates of the tetragonal form of hen egg white lysozyme at 0.1M sodium acetate buffer, pH 4.0, from 4 to 22 C and with 3.0%, 5.0%, and 7.0% NaCl used as the precipitating salt. The data were collected at supersaturation ratios ranging from approximately 4 to approximately 63. Both decreasing temperature and increasing salt concentrations shifted plots of the growth rate versus C/C(sat) to the right, i.e. higher supersaturations were required for comparable growth rates. The observed trends in the growth data are counter to those expected from the solubility data. If tetragonal lysozyme crystal growth is by addition of ordered aggregates from the solution, then the observed growth data could be explained as a result of the effects of lowered temperature and increased salt concentration on the kinetics and equilibrium processes governing protein-protein interactions in solution. The data indicate that temperature would be a more tractable means of controlling the growth rate for tetragonal lysozyme crystals contrary to the usual practice in, e.g., vapor diffusion protein crystal growth, where both the precipitant and protein concentrations are simultaneously increased. However, the available range for control is dependent upon the protein concentration, with the greatest growth rate control being at the lower concentration.

  6. Crystallization of lysozyme with (R)-, (S)- and (RS)-2-methyl-2, 4-pentanediol

    SciTech Connect

    Stauber, Mark; Jakoncic, Jean; Berger, Jacob; Karp, Jerome M.; Axelbaum, Ariel; Sastow, Dahniel; Buldyrev, Sergey V.; Hrnjez, Bruce J.; Asherie, Neer

    2015-03-01

    Crystallization of lysozyme with (R)-2-methyl-2, 4-pentanediol produces more ordered crystals and a higher resolution protein structure than crystallization with (S)-2-methyl-2, 4-pentanediol. The results suggest that chiral interactions with chiral additives are important in protein crystal formation. Chiral control of crystallization has ample precedent in the small-molecule world, but relatively little is known about the role of chirality in protein crystallization. In this study, lysozyme was crystallized in the presence of the chiral additive 2-methyl-2, 4-pentanediol (MPD) separately using the R and S enantiomers as well as with a racemic RS mixture. Crystals grown with (R)-MPD had the most order and produced the highest resolution protein structures. This result is consistent with the observation that in the crystals grown with (R)-MPD and (RS)-MPD the crystal contacts are made by (R)-MPD, demonstrating that there is preferential interaction between lysozyme and this enantiomer. These findings suggest that chiral interactions are important in protein crystallization.

  7. Structural and Mechanistic Studies of Pesticin, a Bacterial Homolog of Phage Lysozymes*

    PubMed Central

    Patzer, Silke I.; Albrecht, Reinhard; Braun, Volkmar; Zeth, Kornelius

    2012-01-01

    Yersinia pestis produces and secretes a toxin named pesticin that kills related bacteria of the same niche. Uptake of the bacteriocin is required for activity in the periplasm leading to hydrolysis of peptidoglycan. To understand the uptake mechanism and to investigate the function of pesticin, we combined crystal structures of the wild type enzyme, active site mutants, and a chimera protein with in vivo and in vitro activity assays. Wild type pesticin comprises an elongated N-terminal translocation domain, the intermediate receptor binding domain, and a C-terminal activity domain with structural analogy to lysozyme homologs. The full-length protein is toxic to bacteria when taken up to the target site via the outer or the inner membrane. Uptake studies of deletion mutants in the translocation domain demonstrate their critical size for import. To further test the plasticity of pesticin during uptake into bacterial cells, the activity domain was replaced by T4 lysozyme. Surprisingly, this replacement resulted in an active chimera protein that is not inhibited by the immunity protein Pim. Activity of pesticin and the chimera protein was blocked through introduction of disulfide bonds, which suggests unfolding as the prerequisite to gain access to the periplasm. Pesticin, a muramidase, was characterized by active site mutations demonstrating a similar but not identical residue pattern in comparison with T4 lysozyme. PMID:22593569

  8. Carboxymethyl chitosan-poly(amidoamine) dendrimer core-shell nanoparticles for intracellular lysozyme delivery.

    PubMed

    Zhang, Xiaoyang; Zhao, Jun; Wen, Yan; Zhu, Chuanshun; Yang, Jun; Yao, Fanglian

    2013-11-06

    Intracellular delivery of native, active proteins is challenging due to the fragility of most proteins. Herein, a novel polymer/protein polyion complex (PIC) nanoparticle with core-shell structure was prepared. Carboxymethyl chitosan-grafted-terminal carboxyl group-poly(amidoamine) (CM-chitosan-PAMAM) dendrimers were synthesized by amidation and saponification reactions. (1)H NMR was used to characterize CM-chitosan-PAMAM dendrimers. The TEM images and results of lysozyme loading efficiency indicated that CM-chitosan-PAMAM dendrimers could self-assemble into core-shell nanoparticles, and lysozyme was efficiently encapsulated inside the core of CM-chitosan-PAMAM dendrimer nanoparticles. Activity of lysozyme was completely inhibited by CM-chitosan-PAMAM Dendrimers at physiological pH, whereas it was released into the medium and exhibited a significant enzymatic activity in an acidic intracellular environment. Moreover, the CM-chitosan-PAMAM dendrimer nanoparticles did not exhibit significant cytotoxicity in the range of concentrations below 3.16 mg/ml. The results indicated that these CM-chitosan-PAMAM dendrimers have excellent properties as highly potent and non-toxic intracellular protein carriers, which would create opportunities for novel applications in protein delivery.

  9. Time-dependent X-ray diffraction studies on urea/hen egg white lysozyme complexes reveal structural changes that indicate onset of denaturation

    PubMed Central

    Raskar, Tushar; Khavnekar, Sagar; Hosur, Madhusoodan

    2016-01-01

    Temporal binding of urea to lysozyme was examined using X-ray diffraction of single crystals of urea/lysozyme complexes prepared by soaking native lysozyme crystals in solutions containing 9 M urea. Four different soak times of 2, 4, 7 and 10 hours were used. The five crystal structures (including the native lysozyme), refined to 1.6 Å resolution, reveal that as the soaking time increased, more and more first-shell water molecules are replaced by urea. The number of hydrogen bonds between urea and the protein is similar to that between protein and water molecules replaced by urea. However, the number of van der Waals contacts to protein from urea is almost double that between the protein and the replaced water. The hydrogen bonding and van der Waals interactions are initially greater with the backbone and later with side chains of charged residues. Urea altered the water-water hydrogen bond network both by replacing water solvating hydrophobic residues and by shortening the first-shell intra-water hydrogen bonds by 0.2 Å. These interaction data suggest that urea uses both ‘direct’ and ‘indirect’ mechanisms to unfold lysozyme. Specific structural changes constitute the first steps in lysozyme unfolding by urea. PMID:27573790

  10. The Shake-and-Bake structure determination of triclinic lysozyme.

    PubMed

    Deacon, A M; Weeks, C M; Miller, R; Ealick, S E

    1998-08-04

    The crystal structure of triclinic lysozyme, comprised of 1,001 non-H protein atoms and approximately 200 bound water molecules, has been determined ab initio (using native data alone) by the "Shake-and-Bake" method by using the computer program SnB. This is the largest structure determined so far by the SnB program. Initial experiments, using default SnB parameters derived from studies of smaller molecules, were unsuccessful. In fact, such experiments produced electron density maps dominated by a single large peak. This problem was overcome by considering the choice of protocol used during the parameter-shift phase refinement. When each phase was subjected to a single shift of +/-157.5 degrees during each SnB cycle, an unusually high percentage of random trials (approximately 22%) yielded correct solutions within 750 cycles. This success rate is higher than that typically observed, even for much smaller structures.

  11. pH dependence of antibody/lysozyme complexation.

    PubMed

    Gibas, C J; Subramaniam, S; McCammon, J A; Braden, B C; Poljak, R J

    1997-12-16

    Association between proteins often depends on the pH and ionic strength conditions of the medium in which it takes place. This is especially true in complexation involving titratable residues at the complex interface. Continuum electrostatics methods were used to calculate the pH-dependent energetics of association of hen egg lysozyme with two closely related monoclonal antibodies raised against it and the association of these antibodies against an avian species variant. A detailed analysis of the energetic contributions reveals that even though the hallmark of association in the two complexes is the presence of conserved charged-residue interactions, the environment of these interactions significantly influences the titration behavior and concomitantly the energetics. The contributing factors include minor structural rearrangements, buried interfacial area, dielectric environment of the key titratable residues, and geometry of the residue dispositions. Modeled structures of several mutant complexes were also studied so as to further delineate the contribution of individual factors to the titration behavior.

  12. Protein Condensation

    NASA Astrophysics Data System (ADS)

    Gunton, James D.; Shiryayev, Andrey; Pagan, Daniel L.

    2007-09-01

    Preface; 1. Introduction; 2. Globular protein structure; 3. Experimental methods; 4. Thermodynamics and statistical mechanics; 5. Protein-protein interactions; 6. Theoretical studies of equilibrium; 7. Nucleation theory; 8. Experimental studies of nucleation; 9. Lysozyme; 10. Some other globular proteins; 11. Membrane proteins; 12. Crystallins and cataracts; 13. Sickle hemoglobin and sickle cell anemia; 14, Alzheimer's disease; Index.

  13. Protein Condensation

    NASA Astrophysics Data System (ADS)

    Gunton, James D.; Shiryayev, Andrey; Pagan, Daniel L.

    2014-07-01

    Preface; 1. Introduction; 2. Globular protein structure; 3. Experimental methods; 4. Thermodynamics and statistical mechanics; 5. Protein-protein interactions; 6. Theoretical studies of equilibrium; 7. Nucleation theory; 8. Experimental studies of nucleation; 9. Lysozyme; 10. Some other globular proteins; 11. Membrane proteins; 12. Crystallins and cataracts; 13. Sickle hemoglobin and sickle cell anemia; 14, Alzheimer's disease; Index.

  14. Enhanced Antibacterial Activity of Acinetobacter baumannii Bacteriophage ØABP-01 Endolysin (LysABP-01) in Combination with Colistin

    PubMed Central

    Thummeepak, Rapee; Kitti, Thawatchai; Kunthalert, Duangkamol; Sitthisak, Sutthirat

    2016-01-01

    Endolysins are lytic enzymes produced by bacteriophages with their ability to degrade the cell wall of bacterial hosts. Endolysin (LysABP-01) from Acinetobacter baumannii bacteriophage ØABP-01 was cloned, overexpressed and characterized. Endolysin LysABP-01 has a globular structure consisting of lysozyme-like (N-acetyl-β-D-muramidase) catalytic domain. It contains 185 amino acids which correspond to a 21.1 kDa protein. The lytic activity of the recombinant endolysin protein was determined by a plate lysis assay for its ability to lyse the autoclaved cell (crude cell wall) of the different bacterial species. LysABP-01 can degrade the crude cell wall of A. baumannii strains, Escherichia coli and Pseudomonas aeruginosa but not of Staphylococcus aureus. The antibacterial activity of LysABP-01 and its synergism with various antibiotics were tested. The results exhibited elevated antibacterial activity in a combination of the sub-MIC LysABP-01 and colistin. The checkerboard assay for measuring antibiotic synergy of LysABP-01 and colistin was performed. This combination was synergistic against various drug-resistant strains of A. baumannii (FIC index < 0.5). In summary, our study highlights the ability of LysABP-01 endolysin to hydrolyze the A. baumannii cell wall and its synergistic interaction with colistin. PMID:27656173

  15. Crystallization of Chicken Egg White Lysozyme from Sulfate Salts

    NASA Technical Reports Server (NTRS)

    Forsythe, Elizabeth; Pusey, Marc

    1998-01-01

    It has been "known" that chicken egg white lysozyme does not crystallize from sulfate, particularly ammonium sulfate, salts, but instead gives amorphous precipitates. This has been the basis of several studies using lysozyme comparing macromolecule crystal nucleation and amorphous precipitation. Recently Ries-Kautt et al (Acta Cryst D50, (1994) 366) have shown that purified isoionic CEWL could be crystallized from low concentrations of sulfate at basic pH, and we subsequently showed that in fact CEWL could be purified in both the tetragonal and orthorhombic forms using ammonium sulfate over the pH range 4.0 to 7.8 (Acta Cryst D53, (1997) 795). We have now extended these observations to include a range of common sulfate salts, specifically sodium, potassium, rubidium, magnesium, and manganese sulfates. In all cases but the manganese sulfates both the familiar tetragonal and orthorhombic forms were obtained, with unit cell dimensions close to those known for the "classic" sodium chloride crystallized forms. Manganese sulfate has only yielded orthorhombic crystals to date. All crystallizations were carried out using low (typically less than or equal to 6 M) salt and high (greater than approximately 90 mg/ml) protein concentrations. As with ammonium sulfate, the tetragonal - orthorhombic phase shift appears to be a function of both the temperature and the protein concentration, with higher temperatures and concentrations favoring the orthorhombic and lower the tetragonal form. The phase change range is somewhat reduced for the sulfate salts, depending upon conditions being typically between approximately 15 - 20 C. Both the magnesium and manganese sulfates gave crystals at salt concentrations over 0.6 M as well, with magnesium sulfate giving a very slowly nucleating and growing hexagonal form. A triclinic crystal form, characterized by aggressively small crystals (typically 0.1 mm in size) has been occasionally obtained from ammonium sulfate. Finally, preliminary spot

  16. Tetragonal Lysozyme Interactions Studied by Site Directed Mutagenesis

    NASA Technical Reports Server (NTRS)

    Crawford, Lisa; Karr, Laurel; Pusey, Marc

    1998-01-01

    A number of recent experimental and theoretical studies have indicated that tetragonal lysozyme crystal growth proceeds by the addition of aggregates, formed by reversible self association of the solute molecules in the bulk'solution. Periodic bond chain and atomic force microscopy studies have indicated that the probable growth unit is at minimum a 43 tetramer, and most likely an octamer composed of two complete turns about the 4(sub 3) axis. If these results are correct, then there are intermolecular interactions which are only formed in the solution and others only formed at the joining of the growth unit to the crystal surface. We have set out to study these interactions, and the correctness of this hypothesis, using site directed mutagenesis of specific amino acid residues involved in the different bonds. We had initially expressed wild type lysozyme in S. cervasiae with yields of approximately 5 mg/L, which were eventually raised to approximately 40 mg/L. We are now moving the expression to the Pichia system, with anticipated yields of 300 to greater than 500 mg/L, comparable to what can be obtained from egg whites. An additional advantage of using recombinant protein is the greater genetic homogeneity of the material obtained and the absence of any other contaminating egg proteins. The first mutation experiments are TYR 23 yields PHE or ALA and ASN 113 yields ALA or ASP. Both TYR 23 and ASN 113 form part of the postulated dimerization intermolecular binding site which lead to the formation of the 4(sub 3) helix. Tyrosine also participates in an intermolecular hydrogen bond with ARG 114. The results of these and subsequent experiments will be discussed.

  17. Tetragonal Lysozyme Interactions Studied by Site Directed Mutagenesis

    NASA Technical Reports Server (NTRS)

    Crawford, Lisa; Karr, Laurel J.; Nadarajah, Arunan; Pusey, Marc

    1999-01-01

    A number of recent experimental and theoretical studies have indicated that tetragonal lysozyme crystal growth proceeds by the addition of aggregates, formed by reversible self association of the solute molecules in the bulk solution. Periodic bond chain and atomic force microscopy studies have indicated that the probable growth unit is at minimum a 43 tetramer, and most likely an octamer composed of two complete turns about the 43 axis. If these results are correct, then there are intermolecular interactions which are only formed in the solution and others only formed at the joining of the growth unit to the crystal surface. We have set out to study these interactions, and the correctness of this hypothesis, using site directed mutagenesis of specific amino acid residues involved in the different bonds. We had initially expressed wild type lysozyme in S. cervasiae with yields of approximately 5 mg/L, which were eventually raised to approximately 40 mg/L. We are now moving the expression to the Pichia system, with anticipated yields of 300 to (3)500 mg/L, comparable to what can be obtained from egg whites. An additional advantage of using recombinant protein is the greater genetic homogeneity of the material obtained and the absence of any other contaminating egg proteins. The first mutation experiments are TYR 23 (Registered) PHE or ALA and ASN 113 (Registered) ALA or ASP. Both TYR 23 and ASN 113 form part of the postulated dimerization intermolecular binding site which lead to the formation of the 43 helix. Tyrosine also participates in an intermolecular hydrogen bond with ARG 114. The results of these and subsequent experiments will be discussed.

  18. Solid-state synthesis and mechanical unfolding of polymers of T4 lysozyme.

    PubMed

    Yang, G; Cecconi, C; Baase, W A; Vetter, I R; Breyer, W A; Haack, J A; Matthews, B W; Dahlquist, F W; Bustamante, C

    2000-01-04

    Recent advances in single molecule manipulation methods offer a novel approach to investigating the protein folding problem. These studies usually are done on molecules that are naturally organized as linear arrays of globular domains. To extend these techniques to study proteins that normally exist as monomers, we have developed a method of synthesizing polymers of protein molecules in the solid state. By introducing cysteines at locations where bacteriophage T4 lysozyme molecules contact each other in a crystal and taking advantage of the alignment provided by the lattice, we have obtained polymers of defined polarity up to 25 molecules long that retain enzymatic activity. These polymers then were manipulated mechanically by using a modified scanning force microscope to characterize the force-induced reversible unfolding of the individual lysozyme molecules. This approach should be general and adaptable to many other proteins with known crystal structures. For T4 lysozyme, the force required to unfold the monomers was 64 +/- 16 pN at the pulling speed used. Refolding occurred within 1 sec of relaxation with an efficiency close to 100%. Analysis of the force versus extension curves suggests that the mechanical unfolding transition follows a two-state model. The unfolding forces determined in 1 M guanidine hydrochloride indicate that in these conditions the activation barrier for unfolding is reduced by 2 kcal/mol.

  19. Mechanistic studies on the release of lysozyme from twin-screw extruded lipid implants.

    PubMed

    Sax, Gerhard; Winter, Gerhard

    2012-10-28

    The influence of lipid melting on the in-vitro release of lysozyme from twin-screw extruded lipid implants was investigated. Triglyceride based implants were prepared by admixing of glycerol tristearin and various low melting lipids and subsequent twin-screw extrusion (tsc-extrusion) of these mixtures at moderate temperatures. Lysozyme was embedded as model protein and PEG 4000 or PEG 6000 was used as pore-forming excipient. By decreasing the amount of pore-forming agent from 40% to 0% lysozyme release became more sustained and the release kinetics changed from a matrix-type release profile to a linear release profile. Differential scanning calorimetry, X-ray diffraction and scanning electron microscopy measurements showed a change in implant structure upon long-term release (240 days) at 37 °C which was explained by partial matrix melting. In addition, partial melting of the implants was found to facilitate complete drug release at 37 °C whereas at 20 °C without partial melting 20% to 90% of the incorporated protein remained trapped in the implant matrix. In conclusion, partial melting of the implants during in-vitro release was found to be a major factor for the control of protein release from extruded implants and can be useful to trigger release, achieve in-vivo biodegradability and complete long-term protein release.

  20. Multiple specialised goose-type lysozymes potentially compensate for an exceptional lack of chicken-type lysozymes in Atlantic cod.

    PubMed

    Seppola, Marit; Bakkemo, Kathrine Ryvold; Mikkelsen, Helene; Myrnes, Bjørnar; Helland, Ronny; Irwin, David M; Nilsen, Inge W

    2016-06-21

    Previous analyses of the Atlantic cod genome showed unique combinations of lacking and expanded number of genes for the immune system. The present study examined lysozyme activity, lysozyme gene distribution and expression in cod. Enzymatic assays employing specific bacterial lysozyme inhibitors provided evidence for presence of g-type, but unexpectedly not for c-type lysozyme activity. Database homology searches failed to identify any c-type lysozyme gene in the cod genome or in expressed sequence tags from cod. In contrast, we identified four g-type lysozyme genes (LygF1a-d) constitutively expressed, although differentially, in all cod organs examined. The active site glutamate residue is replaced by alanine in LygF1a, thus making it enzymatic inactive, while LygF1d was found in two active site variants carrying alanine or glutamate, respectively. In vitro and in vivo infection by the intracellular bacterium Francisella noatunensis gave a significantly reduced LygF1a and b expression but increased expression of the LygF1c and d genes as did also the interferon gamma (IFNγ) cytokine. These results demonstrate a lack of c-type lysozyme that is unprecedented among vertebrates. Our results further indicate that serial gene duplications have produced multiple differentially regulated cod g-type lysozymes with specialised functions potentially compensating for the lack of c-type lysozymes.

  1. Crystallization of Chicken Egg-White Lysozyme from Ammonium Sulfate

    NASA Technical Reports Server (NTRS)

    Forsythe, Elizabeth L.; Snell, Edward H.; Pusey, Marc L.

    1997-01-01

    Chicken egg-white lysozyme was crystallized from ammonium sulfate over the pH range 4.0-7.8, with protein concentrations from 100 to 150 mg/ml. Crystals were obtained by vapor-diffusion or batch-crystallization methods. The protein crystallized in two morphologies with an apparent morphology dependence on temperature and protein concentration. In general, tetragonal crystals could be grown by lowering the protein concentration or temperature. Increasing the temperature or protein concentration resulted in the growth of orthorhombic crystals. Representative crystals of each morphology were selected for X-ray analysis. The tetragonal crystals belonged to the P4(sub 3)2(sub 1)2 space group with crystals grown at ph 4.4 having unit-cell dimensions of a = b = 78.7 1, c=38.6 A and diffracting to beyond 2.0 A. The orthorhombic crystals, grown at pH 4.8, were of space group P2(sub 1)2(sub 1)2 and had unit-cell dimensions of a = 30.51, b = 56.51 and c = 73.62 A.

  2. Low Viscosity Highly Concentrated Injectable Nonaqueous Suspensions of Lysozyme Microparticles

    PubMed Central

    Miller, Maria A.; Engstrom, Joshua D.; Ludher, Baltej S.; Johnston, Keith P.

    2011-01-01

    Subcutaneous injection of concentrated protein and peptide solutions, in the range of 100–400 mg/mL, is often not possible with a 25- to 27-gauge needle, as the viscosity can be well above 50 cP. Apparent viscosities below this limit are reported for suspensions of milled lysozyme microparticles up to nearly 400 mg/mL in benzyl benzoate or benzyl benzoate mixtures with safflower oils through a syringe with a 25- to 27-gauge needle at room temperature. These apparent viscosities were confirmed using a cone-and-plate rheometer. The intrinsic viscosity regressed from the Kreiger–Dougherty model was only slightly above the Einstein value of 2.5, indicating the increase in viscosity relative to that of the solvent was caused primarily by the excluded volume. Thus, the increases in viscosity from electrical double layer interactions (electroviscous effects), solvation of the particles, or deviations of the particle shape from a spherical geometry were minimal, and much smaller than typically observed for proteins dissolved in aqueous solutions. The small electroviscous effects are expected given the negligible zeta potential and thin double layers in the low dielectric constant organic solvent. The suspensions were resuspendable after a year, with essentially constant particle size after two months as measured by static light scattering. The lower apparent viscosities for highly concentrated protein suspensions relative to protein solutions, coupled with these favorable characteristics upon resuspension, may offer novel opportunities for subcutaneous injection of therapeutic proteins. PMID:19803503

  3. Detection of recombinant human lactoferrin and lysozyme produced in a bitransgenic cow.

    PubMed

    Kaiser, Germán G; Mucci, Nicolás C; González, Vega; Sánchez, Lourdes; Parrón, José A; Pérez, María D; Calvo, Miguel; Aller, Juan F; Hozbor, Federico A; Mutto, Adrián A

    2017-03-01

    Lactoferrin and lysozyme are 2 glycoproteins with great antimicrobial activity, being part of the nonspecific defensive system of human milk, though their use in commercial products is difficult because human milk is a limited source. Therefore, many investigations have been carried out to produce those proteins in biological systems, such as bacteria, yeasts, or plants. Mammals seem to be more suitable as expression systems for human proteins, however, especially for those that are glycosylated. In the present study, we developed a bicistronic commercial vector containing a goat β-casein promoter and an internal ribosome entry site fragment between the human lactoferrin and human lysozyme genes to allow the introduction of both genes into bovine adult fibroblasts in a single transfection. Embryos were obtained by somatic cell nuclear transfer, and, after 6 transferences to recipients, 3 pregnancies and 1 viable bitransgenic calf were obtained. The presence of the vector was confirmed by fluorescent in situ hybridization of skin cells. At 13 mo of life and after artificial induction of lactation, both recombinant proteins were found in the colostrum and milk of the bitransgenic calf. Human lactoferrin concentration in the colostrum was 0.0098 mg/mL and that in milk was 0.011 mg/mL; human lysozyme concentration in the colostrum was 0.0022 mg/mL and that in milk was 0.0024 mg/mL. The molar concentration of both human proteins revealed no differences in protein production of the internal ribosome entry site upstream and downstream protein. The enzymatic activity of lysozyme in the transgenic milk was comparable to that of human milk, being 6 and 10 times higher than that of bovine lysozyme present in milk. This work represents an important step to obtain multiple proteins or enhance single protein production by using animal pharming and fewer regulatory and antibiotic-resistant foreign sequences, allowing the design of humanized milk with added biological value for

  4. Modeling the Growth Rates of Tetragonal Lysozyme Crystal Faces

    NASA Technical Reports Server (NTRS)

    Li, Meirong; Nadarajah, Arunan; Pusey, Marc L.

    1998-01-01

    with respect to its concentration at saturation in order to apply growth rate models to this process. The measured growth rates were then compared with the predicted ones from several dislocation and 2D nucleation growth models, employing tetramer and octamer growth units in polydisperse solutions and monomer units in monodisperse solutions. For the (110) face, the calculations consistently showed that the measured growth rates followed the expected model relations with octamer growth units. For the (101) face, it is not possible to obtain a clear agreement between the predicted and measured growth rates for a single growth unit as done for the (110) face. However, the calculations do indicate that the average size of the growth unit is between a tetramer and an octamer. This suggests that tetramers, octamers and other intermediate size growth units all participate in the growth process for this face. These calculations show that it is possible to model the macroscopic protein crystal growth rates if the molecular level processes can be account for, particularly protein aggregation processes in the bulk solution. Our recent investigations of tetragonal lysozyme crystals employing high resolution atomic force microscopy scans have further confirmed the growth of these crystals by aggregate growth units corresponding to 4(sub 3) helices.

  5. Anti-fibrillation propensity of a flavonoid baicalein against the fibrils of hen egg white lysozyme: potential therapeutics for lysozyme amyloidosis.

    PubMed

    Fazili, Naveed Ahmad; Bhat, Imtiyaz Ahmad; Bhat, Waseem Feeroze; Naeem, Aabgeena

    2016-10-01

    More than 20 human diseases involve the fibrillation of a specific protein/peptide which forms pathological deposits at various sites. Hereditary lysozyme amyloidosis is a systemic disorder which mostly affects liver, spleen and kidney. This conformational disorder is featured by lysozyme fibril formation. In vivo lysozyme fibrillation was simulated under in vitro conditions using a strong denaturant GdHCl at 3 M concentration. Sharp decline in the ANS fluorescence intensity compared to the partially unfolded states, almost 20-fold increase in ThT fluorescence intensity, increase in absorbance at 450 nm suggesting turbidity, negative ellipticity peak in the far-UVCD at 217 nm, red shift of 50 nm compared to the native state in Congo red assay and appearance of a network of long rope-like fibrils in transmission electron microscope (TEM) analysis suggested HEWL fibrillation. Anti-fibrillation potency of baicalein against the preformed fibrils of HEWL was investigated following ThT assay in which there was a dose-dependent decrease in ThT fluorescence intensity compared to the fibrillar state of HEWL with the maximum effect observed at 150-μM baicalein concentration, loss of negative ellipticity peak in the far-UVCD region, dip in the Rayleigh scattering intensity and absorbance at 350 and 450 nm, respectively, together with a reduction in the density of fibrillar structure in TEM imaging. Thus, it could be suggested that baicalein could prove to be a positive therapeutics for hereditary human lysozyme amyloidosis.

  6. The preparation of protected fragments of lysozyme for semisynthesis.

    PubMed Central

    Rees, A R; Offord, R E

    1976-01-01

    This paper reports the development of methods for preparing tryptic fragments of hen's-egg lysozyme in an appropriate state of protection for use in the chemical synthesis of modified polypeptides. 1. We describe the cleavage of the disulphide bridges of the enzyme and the simulatneous protection of the liberated thiol groups by S-sulphonation. Lysozyme resisted the usual conditions for this reaction. We have confirmed the stability of the S-sulphonyl group to the conditions met in peptide synthesis. 2. We describe the reversible protection of the amino groups of the enzyme by reaction with various anhydrides of 1,2-dicarboxylic acids. We conclude that 2-methylmaleic anhydride and exo-cis-3,6-endoxo-delta4-tetrahydrophthalic anhydride are unsuitable for our purpose but that maleic anhydride can, in spite of certain drawbacks, be used. 3. We describe the tryptic cleavage of the thiol- and amino-protected protein and the separation of the fragments. 4. We describe the reversible protection of the carboxylic acid groups (including the specific deprotection of the alpha-carboxyl group), the imidazolyl group and the aloph-amino groups of the fragments. Several alternative groups have been evaluated for most of these purposes. The side-chain amides did not present any serious problem of libility, 5. We describe experiments on the stability of the side chain of tryptophan, both protected by formylation and unprotected, to the acid conditions needed for the deprotection of the other functional groups in the peptide. We conclude that protection of tryptophan is unnecessary. We suggest that most of the methods described are of general application in peptide semisynthesis by fragment condensation. An Appendix is included to which points 6-ll appertain... PMID:1008811

  7. Ortho-methylated 3-hydroxypyridines hinder hen egg-white lysozyme fibrillogenesis

    PubMed Central

    Mariño, Laura; Pauwels, Kris; Casasnovas, Rodrigo; Sanchis, Pilar; Vilanova, Bartolomé; Muñoz, Francisco; Donoso, Josefa; Adrover, Miquel

    2015-01-01

    Protein aggregation with the concomitant formation of amyloid fibrils is related to several neurodegenerative diseases, but also to non-neuropathic amyloidogenic diseases and non-neurophatic systemic amyloidosis. Lysozyme is the protein involved in the latter, and it is widely used as a model system to study the mechanisms underlying fibril formation and its inhibition. Several phenolic compounds have been reported as inhibitors of fibril formation. However, the anti-aggregating capacity of other heteroaromatic compounds has not been studied in any depth. We have screened the capacity of eleven different hydroxypyridines to affect the acid-induced fibrillization of hen lysozyme. Although most of the tested hydroxypyridines alter the fibrillation kinetics of HEWL, only 3-hydroxy-2-methylpyridine, 3-hydroxy-6-methylpyridine and 3-hydroxy-2,6-dimethylpyridine completely abolish fibril formation. Different biophysical techniques and several theoretical approaches are combined to elucidate their mechanism of action. O-methylated 3-hydroxypyridines bind non-cooperatively to two distinct but amyloidogenic regions of monomeric lysozyme. This stabilises the protein structure, as evidenced by enhanced thermal stability, and results in the inhibition of the conformational transition that precedes fibril assembly. Our results point to o-methylated 3-hydroxypyridines as a promising molecular scaffold for the future development of novel fibrillization inhibitors. PMID:26169912

  8. Ortho-methylated 3-hydroxypyridines hinder hen egg-white lysozyme fibrillogenesis

    NASA Astrophysics Data System (ADS)

    Mariño, Laura; Pauwels, Kris; Casasnovas, Rodrigo; Sanchis, Pilar; Vilanova, Bartolomé; Muñoz, Francisco; Donoso, Josefa; Adrover, Miquel

    2015-07-01

    Protein aggregation with the concomitant formation of amyloid fibrils is related to several neurodegenerative diseases, but also to non-neuropathic amyloidogenic diseases and non-neurophatic systemic amyloidosis. Lysozyme is the protein involved in the latter, and it is widely used as a model system to study the mechanisms underlying fibril formation and its inhibition. Several phenolic compounds have been reported as inhibitors of fibril formation. However, the anti-aggregating capacity of other heteroaromatic compounds has not been studied in any depth. We have screened the capacity of eleven different hydroxypyridines to affect the acid-induced fibrillization of hen lysozyme. Although most of the tested hydroxypyridines alter the fibrillation kinetics of HEWL, only 3-hydroxy-2-methylpyridine, 3-hydroxy-6-methylpyridine and 3-hydroxy-2,6-dimethylpyridine completely abolish fibril formation. Different biophysical techniques and several theoretical approaches are combined to elucidate their mechanism of action. O-methylated 3-hydroxypyridines bind non-cooperatively to two distinct but amyloidogenic regions of monomeric lysozyme. This stabilises the protein structure, as evidenced by enhanced thermal stability, and results in the inhibition of the conformational transition that precedes fibril assembly. Our results point to o-methylated 3-hydroxypyridines as a promising molecular scaffold for the future development of novel fibrillization inhibitors.

  9. Immobilization of lysozyme on cotton fabrics; synthesis, characterication, and activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The antimicrobial activity of lysozyme derives from the hydrolysis of the bacterial cell wall polysaccharide at the glycosidic bond that links N-acetyl-glucosamine and N-acetyl-muramic acid. Maintaining the activity of lysozyme while bound to a cellulose substrate is a goal toward developing enzyme...

  10. Regenerated cellulose fiber and film immobilized with lysozyme

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The present work reports an initial engineering approach for fabricating lysozyme-bound regenerated cellulose fiber and film. Glycine-esterified cotton was dissolved in an ionic liquid solvent 1–Butyl–3–methylimidazolium Chloride (BMIMCl) in which lysozyme was activated and covalently attached to c...

  11. Purification of Lysozyme by Intrinsically Shielded Hydrogel Beads

    NASA Astrophysics Data System (ADS)

    Li, Cong; Zhang, R.; Wang, L.; Bowyer, A.; Eisenthal, R.; Shen, Yehua; Hubble, J.

    2013-07-01

    Macro-sized intrinsically shielded hydrogel beads have been prepared from BSA and CM-dextran grafted with CB using a technique based on freeze-thawing gelation method. The size of the beads lies in around 500 μm. Isothemal titration calorimetry (ITC) showed that the relative binding affinities of the lysozyme for CB, compared with BSA, at pH 3.0 was stronger than that at pH 7.4. They were employed for the affinity separation of lysozyme using chromatography column. Their adsorption capacity for lysozyme at pH 3.0 is higher than that at pH 9. In a binary mixture of lysozyme and ovalbumin, the beads showed very high selectivity toward lysozyme. Lysozyme of very high purity (> 93%) was obtained from a mixture of lysozyme and ovalbumin, and 85% from egg white solution. The results indicate that the macro-sized bead can be used for the separation, purification, and recovery of lysozyme in a chromatograph column.

  12. Crystallization of Chicken Egg White Lysozyme from Assorted Sulfate Salts

    NASA Technical Reports Server (NTRS)

    Forsythe, Elizabeth L.; Snell, Edward H.; Malone, Christine C.; Pusey, Marc L.

    1998-01-01

    Chicken egg white lysozyme has been found to crystallize from ammonium, sodium, potassium, rubidium, magnesium, and manganese sulfates at acidic and basic pH, with protein concentrations from 60 to 190 mg/ml. Four different crystal morphologies have been obtained, depending upon the temperature, protein concentration, and precipitating salt employed, Crystals grown at 15 C were generally tetragonal, with space group P43212. Crystallization at 20 C typically resulted in the formation of orthorhombic crystals, space group P21212 1. The tetragonal much less than orthorhombic morphology transition appeared to be a function of both the temperature and protein concentration, occurring between 15 and 20 C and between 100 and 125 mg/ml protein concentration. Crystallization from 0.8 -1.2M magnesium sulfate at pH 7.6 - 8.0 gave a hexagonal (trigonal) crystal form, space group P3121, which diffracted to 2.8 A. Ammonium sulfate was also found to result in a monoclinic form, space group C2. Small twinned monoclinic crystals of approx. 0.2 mm on edge were grown by dialysis followed by seeded sitting drop crystallization.

  13. Molecular dynamics simulation of thionated hen egg white lysozyme.

    PubMed

    Huang, Wei; Eichenberger, Andreas P; van Gunsteren, Wilfred F

    2012-08-01

    Understanding of the driving forces of protein folding is a complex challenge because different types of interactions play a varying role. To investigate the role of hydrogen bonding involving the backbone, the effect of thio substitutions in a protein, hen egg white lysozyme (HEWL), was investigated through molecular dynamics simulations of native as well as partly (only residues in loops) and fully thionated HEWL using the GROMOS 54A7 force field. The results of the three simulations show that the structural properties of fully thionated HEWL clearly differ from those of the native protein, while for partly thionated HEWL they only changed slightly compared with native HEWL. The analysis of the torsional-angle distributions and hydrogen bonds in the backbone suggests that the α-helical segments of native HEWL tend to show a propensity to convert to 3(10)-helical geometry in fully thionated HEWL. A comparison of the simulated quantities with experimental NMR data such as nuclear overhauser effect (NOE) atom-atom distance bounds and (3)J((H)(N)(H)(α))-couplings measured for native HEWL illustrates that the information content of these quantities with respect to the structural changes induced by thionation of the protein backbone is rather limited.

  14. Molecular dynamics simulation of thionated hen egg white lysozyme

    PubMed Central

    Huang, Wei; Eichenberger, Andreas P; van Gunsteren, Wilfred F

    2012-01-01

    Understanding of the driving forces of protein folding is a complex challenge because different types of interactions play a varying role. To investigate the role of hydrogen bonding involving the backbone, the effect of thio substitutions in a protein, hen egg white lysozyme (HEWL), was investigated through molecular dynamics simulations of native as well as partly (only residues in loops) and fully thionated HEWL using the GROMOS 54A7 force field. The results of the three simulations show that the structural properties of fully thionated HEWL clearly differ from those of the native protein, while for partly thionated HEWL they only changed slightly compared with native HEWL. The analysis of the torsional-angle distributions and hydrogen bonds in the backbone suggests that the α-helical segments of native HEWL tend to show a propensity to convert to 310-helical geometry in fully thionated HEWL. A comparison of the simulated quantities with experimental NMR data such as nuclear overhauser effect (NOE) atom–atom distance bounds and 3JHNHα-couplings measured for native HEWL illustrates that the information content of these quantities with respect to the structural changes induced by thionation of the protein backbone is rather limited. PMID:22653637

  15. Dimer formation in radiation-irradiated aqueous solution of lysozyme studied by light-scattering-intensity measurement.

    PubMed

    Hashimoto, S; Seki, H; Masuda, T; Imamura, M; Kondo, M

    1981-07-01

    The reaction of lysozyme with OH., Br.-2 and e-aq, produced in an aqueous solution by pulsed electrons and gamma-rays, were investigated. Irradiated enzymes showed an increase in the light scattering intensity (LSI) which is proportional to the absorbed dose. Results obtained from SDS gel electrophoresis confirm dimerization of lysozyme, which is considered to be responsible for the increase in LSI. It was found that the rate constant of the dimerization of protein radicals produced in the reaction with OH. is 2K=(1.0 +/- 0.3) X 10(6)M-1 s-1 and the yield of the dimerization is 0.6 in G. The enzymatic activity of the dimer is shown to be reduced to about 30 per cent of that of the intact enzyme. It is concluded that the radiation-induced inactivation of lysozyme is largely due to dimerization.

  16. An experimental model of affinity filtration for the isolation of egg white Lysozyme using Cibacron Blue immobilized to yeast cells.

    PubMed

    Ferraris, María del Pilar; Gonzalez, Ulises A; Aguilar, Carlos F; Rodríguez, Jorge A

    2016-05-01

    An experimental model of affinity filtration process was designed using a macroligand composed by Cibacron Blue F3GA immobilized to yeast cells. Its performance was evaluated, at bench scale, through the recovery of egg white Lysozyme. The selective and reversible binding between the Cibacron ligand molecule and the enzyme is described. The separation of Lysozyme from the protein mixture included the application of stages such as affinity adsorption, concentration, diafiltration and elution. A tangential microfiltration system with an inorganic membrane was designed. The main finding was the development of the diafiltration operation, key stage in the enzyme isolation. The macroligand particle kept its integrity along the whole process and the degree of purity of the isolated Lysozyme was significant.

  17. Anti-protozoal and anti-bacterial antibiotics that inhibit protein synthesis kill cancer subtypes enriched for stem cell-like properties.

    PubMed

    Cuyàs, Elisabet; Martin-Castillo, Begoña; Corominas-Faja, Bruna; Massaguer, Anna; Bosch-Barrera, Joaquim; Menendez, Javier A

    2015-01-01

    Key players in translational regulation such as ribosomes might represent powerful, but hitherto largely unexplored, targets to eliminate drug-refractory cancer stem cells (CSCs). A recent study by the Lisanti group has documented how puromycin, an old antibiotic derived from Streptomyces alboniger that inhibits ribosomal protein translation, can efficiently suppress CSC states in tumorspheres and monolayer cultures. We have used a closely related approach based on Biolog Phenotype Microarrays (PM), which contain tens of lyophilized antimicrobial drugs, to assess the chemosensitivity profiles of breast cancer cell lines enriched for stem cell-like properties. Antibiotics directly targeting active sites of the ribosome including emetine, puromycin and cycloheximide, inhibitors of ribosome biogenesis such as dactinomycin, ribotoxic stress agents such as daunorubicin, and indirect inhibitors of protein synthesis such as acriflavine, had the largest cytotoxic impact against claudin-low and basal-like breast cancer cells. Thus, biologically aggressive, treatment-resistant breast cancer subtypes enriched for stem cell-like properties exhibit exacerbated chemosensitivities to anti-protozoal and anti-bacterial antibiotics targeting protein synthesis. These results suggest that old/existing microbicides might be repurposed not only as new cancer therapeutics, but also might provide the tools and molecular understanding needed to develop second-generation inhibitors of ribosomal translation to eradicate CSC traits in tumor tissues.

  18. Epigenetic Activation of Antibacterial Property of an Endophytic Streptomyces coelicolor Strain AZRA 37 and Identification of the Induced Protein Using MALDI TOF MS/MS

    PubMed Central

    Kumar, Jitendra; Sharma, Vijay K.; Singh, Dheeraj K.; Mishra, Ashish; Gond, Surendra K.; Verma, Satish K.; Kumar, Anuj; Kharwar, Ravindra Nath

    2016-01-01

    The endophytic Streptomyces coelicolor strain AZRA 37 was isolated from the surface sterilized root of Azadirachta indica A. Juss., commonly known as neem plant in India. Since only a few reports are available regarding epigenetic modulations of microbial entities, S. coelicolor was treated with different concentrations of 5-azacytidine for this purpose and evaluated for its antibacterial potential against five human pathogenic bacteria (Aeromonas hydrophila IMS/GN11, Enterococcus faecalis IMS/GN7, Salmonella typhi MTCC 3216, Shigella flexneri ATCC 12022 and Staphylococcus aureus ATCC 25923). The crude extract obtained from cultures treated with 25 μM concentration of 5-azacytidine, was found effective against all five pathogenic bacteria tested while the untreated control was only active against 3 pathogenic bacteria. HPLC analysis of crude compounds from treated cultures showed a greater number of compounds than that of the control. Extraction of whole cell protein and its SDS PAGE analysis showed an additional major protein band in 25 μM 5-azacytidine treated culture and MALDI TOF MS/MS analysis revealed that this protein belongs to the porin family. PMID:26844762

  19. Immobilization of lysozyme-cellulose amide-linked conjugates on cellulose I and II cotton nanocrystalline preparations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lysozyme was attached through an amide linkage between protein aspartate and glutamate residues to amino-glycine-cellulose (AGC), which was prepared by esterification of glycine to preparations of cotton nanocrystals (CNC). The nanocrystalline preparations were produced through acid hydrolysis and ...

  20. Microcalorimetric study of thermal unfolding of lysozyme in water/glycerol mixtures: An analysis by solvent exchange model

    NASA Astrophysics Data System (ADS)

    Spinozzi, Francesco; Ortore, Maria Grazia; Sinibaldi, Raffaele; Mariani, Paolo; Esposito, Alessandro; Cinelli, Stefania; Onori, Giuseppe

    2008-07-01

    Folded protein stabilization or destabilization induced by cosolvent in mixed aqueous solutions has been studied by differential scanning microcalorimetry and related to difference in preferential solvation of native and denatured states. In particular, the thermal denaturation of a model system formed by lysozyme dissolved in water in the presence of the stabilizing cosolvent glycerol has been considered. Transition temperatures and enthalpies, heat capacity, and standard free energy changes have been determined when applying a two-state denaturation model to microcalorimetric data. Thermodynamic parameters show an unexpected, not linear, trend as a function of solvent composition; in particular, the lysozyme thermodynamic stability shows a maximum centered at water molar fraction of about 0.6. Using a thermodynamic hydration model based on the exchange equilibrium between glycerol and water molecules from the protein solvation layer to the bulk, the contribution of protein-solvent interactions to the unfolding free energy and the changes of this contribution with solvent composition have been derived. The preferential solvation data indicate that lysozyme unfolding involves an increase in the solvation surface, with a small reduction of the protein-preferential hydration. Moreover, the derived changes in the excess solvation numbers at denaturation show that only few solvent molecules are responsible for the variation of lysozyme stability in relation to the solvent composition.

  1. Molecular dynamics simulation of lysozyme adsorption/desorption on hydrophobic surfaces.

    PubMed

    Wei, Tao; Carignano, Marcelo A; Szleifer, Igal

    2012-08-30

    In this work, we present a series of fully atomistic molecular dynamics (MD) simulations to study lysozyme's orientation-dependent adsorption on polyethylene (PE) surface in explicit water. The simulations show that depending on the orientation of the initial approach to the surface the protein may adsorb or bounce from the surface. The protein may completely leave the surface or reorient and approach the surface resulting in adsorption. The success of the trajectory to adsorb on the surface is the result of different competing interactions, including protein-surface interactions and the hydration of the protein and the hydrophobic PE surface. The difference in the hydration of various protein sites affects the protein's orientation-dependent behavior. Side-on orientation is most likely to result in adsorption as the protein-surface exhibits the strongest attraction. However, adsorption can also happen when lysozyme's longest axis is tilted on the surface if the protein-surface interaction is large enough to overcome the energy barrier that results from dehydrating both the protein and the surface. Our study demonstrates the significant role of dehydration process on hydrophobic surface during protein adsorption.

  2. Lysozyme entrapped within reverse hexagonal mesophases: physical properties and structural behavior.

    PubMed

    Mishraki, Tehila; Libster, Dima; Aserin, Abraham; Garti, Nissim

    2010-01-01

    A model protein (lysozyme) was incorporated into monoolein-based reverse hexagonal (H(II)) mesophase and its structure effects were characterized by small angle X-ray scattering, ATR-FTIR spectroscopy, and rheological measurements. Modifications in molecular organization of the H(II) mesophases as well as the conformational stability of lysozyme (LSZ) as a function of pH and denaturating agent (urea) were clarified. Up to 3 wt.% LSZ can be solubilized into the H(II). The vibration FTIR analysis revealed that LSZ interacted with OH groups of glycerol monooleate (GMO) in the outer interface region, resulting in strong hydrogen bonding between the surfactant and its environment. Simultaneously, the decrease in the hydrogen-bonded carbonyl population of GMO was monitored, indicating dehydration of the monoolein carbonyls. These molecular interactions yielded a minor decrease in the lattice parameter of the systems, as detected by small angle X-ray scattering. Furthermore, LSZ was crystallized within the medium of the hexagonal structures in a single crystal form. The alpha-helix conformation of lysozyme was stabilized at high pH conditions, demonstrating greater helical structure content, compared to D(2)O solution. Moreover, the hexagonal phase decreased the unfavorable alpha-->beta transition in lysozyme, thereby increasing the stability of the protein under chemical denaturation. The rheological behavior of the hexagonal structures varied with the incorporation of LSZ, reflected in stronger elastic properties and pronounced solid-like response of the systems. The hydrogen bonding enhancement in the interface region of the structures was most likely responsible for these phenomena. The results of this study provided valuable information on the use of hexagonal systems as a carrier for incorporation and stabilization of proteins for various applications.

  3. Lysozyme as an alternative to antibiotics improves performance in nursery pigs during an indirect immune challenge.

    PubMed

    Oliver, W T; Wells, J E; Maxwell, C V

    2014-11-01

    Lysozyme is a 1,4-β-N-acetylmuramidase that has antimicrobial properties. The objective of this study was to determine the effect of lysozyme and antibiotics on growth performance and immune response during an indirect immune challenge. Two replicates of 600 pigs each were weaned from the sow at 26 d of age, blocked by litter and sex, and then randomly assigned to 1 of 24 pens in either a nursery room that had been fully disinfected or a nursery room left unclean since the previous group of pigs. Within a room, pigs were randomly assigned to either control diets (2 phase nursery regime), control diets + antibiotics (chlortetracycline/tiamulin hydrogen fumarate), or control diets + lysozyme (100 mg/kg diet). Pig weights and feed disappearance were measured and blood was collected on d 0, 14, and 28 of treatment. A group of 20 pigs were killed at 24 d of age for initial body composition analysis and 10 pigs of median weight were killed per diet room combination for body composition analysis after 28 d of treatment. Control + antibiotics and control + lysozyme-fed pigs grew at a faster rate for the 28-d study compared to control pigs (318 ± 14,320 ± 15 vs. 288 ± 15 g/d, respectively; P < 0.05), regardless of nursery environment (P > 0.05). The indirect immune challenge did not alter growth performance from d 0 to 14 of treatment but decreased ADG from d 14 to 28 of the study (415 ± 15 vs. 445 ± 13 g/d ;: P < 0.05). Feed intake was not altered by the nursery environment (P > 0.61) or dietary treatments (P > 0.10), but feed efficiency was worsened by the indirect immune challenge (P < 0.05) and improved by both control + antibiotics and control + lysozyme diets (P < 0.01). The immune challenge did not alter nutrient accretion (P > 0.25), but both control + antibiotics and control + lysozyme pigs had decreased accretion of whole-body lipid (P < 0.01) and increased accretion of protein (P < 0.09). Blood levels of tumor necrosis factor-α (TNF-α; P < 0

  4. Effect of PEG molecular weight and PEGylation degree on the physical stability of PEGylated lysozyme.

    PubMed

    Morgenstern, Josefine; Baumann, Pascal; Brunner, Carina; Hubbuch, Jürgen

    2017-03-15

    During production, purification, formulation, and storage proteins for pharmaceutical or biotechnological applications face solution conditions that are unfavorable for their stability. Such harmful conditions include extreme pH changes, high ionic strengths or elevated temperatures. The characterization of the main influencing factors promoting undesired changes of protein conformation and aggregation, as well as the manipulation and selective control of protein stabilities are crucially important to biopharmaceutical research and process development. In this context PEGylation, i.e. the covalent attachment of polyethylene glycol (PEG) to proteins, represents a valuable strategy to improve the physico-chemical properties of proteins. In this work, the influence of PEG molecular weight and PEGylation degree on the physical stability of PEGylated lysozyme is investigated. Specifically, conformational and colloidal properties were studied by means of high-throughput melting point determination and automated generation of protein phase diagrams, respectively. Lysozyme from chicken egg-white as a model protein was randomly conjugated to 2kDa, 5kDa and 10kDa mPEG-aldehyde and resulting PEGamer species were purified by chromatographic separation. Besides protein stability assessment, residual enzyme activities were evaluated employing a Micrococcus lysodeikticus based activity assay. PEG molecules with lower molecular weights and lower PEGylation degrees resulted in higher residual activities. Changes in enzyme activities upon PEGylation have shown to result from a combination of steric hindrance and molecular flexibility. In contrast, higher PEG molecular weights and PEGylation degrees enhanced conformational and colloidal stability. By PEGylating lysozyme an increase of the protein solubility by more than 11-fold was achieved.

  5. Antibacterial gene transfer across the tree of life

    PubMed Central

    Metcalf, Jason A; Funkhouser-Jones, Lisa J; Brileya, Kristen; Reysenbach, Anna-Louise; Bordenstein, Seth R

    2014-01-01

    Though horizontal gene transfer (HGT) is widespread, genes and taxa experience biased rates of transferability. Curiously, independent transmission of homologous DNA to archaea, bacteria, eukaryotes, and viruses is extremely rare and often defies ecological and functional explanations. Here, we demonstrate that a bacterial lysozyme family integrated independently in all domains of life across diverse environments, generating the only glycosyl hydrolase 25 muramidases in plants and archaea. During coculture of a hydrothermal vent archaeon with a bacterial competitor, muramidase transcription is upregulated. Moreover, recombinant lysozyme exhibits broad-spectrum antibacterial action in a dose-dependent manner. Similar to bacterial transfer of antibiotic resistance genes, transfer of a potent antibacterial gene across the universal tree seemingly bestows a niche-transcending adaptation that trumps the barriers against parallel HGT to all domains. The discoveries also comprise the first characterization of an antibacterial gene in archaea and support the pursuit of antibiotics in this underexplored group. DOI: http://dx.doi.org/10.7554/eLife.04266.001 PMID:25422936

  6. Functional analysis of two invertebrate-type lysozymes from red swamp crayfish, Procambarus clarkii.

    PubMed

    Zhang, Hong-Wei; Sun, Chen; Sun, Shan-Shan; Zhao, Xiao-Fan; Wang, Jin-Xing

    2010-12-01

    Lysozyme is an important immune effector and is widely distributed in many organisms. In the present study, two novel invertebrate-type lysozymes (Pclysi1 and Pclysi2) were cloned from red swamp crayfish, Procambarus clarkii. Alignment analysis showed that these two genes were different in catalytic residues. Results of RT-PCR showed that these two genes share similar tissue distribution patterns, and both were upregulated after bacteria challenge. The mature recombinant proteins were expressed in Escherichia coli system and purified. Activity analysis revealed that rPclysi1 had no muramidase activity and isopeptidase activity, but had antimicrobial activity. Meanwhile, rPclysi2 was muramidase-deficient and had no antimicrobial activity, but possessed isopeptidase activity. Above data suggest that Pclysi1 and 2 may offer different functions in the crayfish immunity.

  7. [Relation between the lysozyme hydration isotherm and molecule packing in the solid phase].

    PubMed

    Gevorkian, S G; Morozov, V N

    1983-01-01

    A micromethod for measurement of mass changes of glutaraldehyde treated protein crystals is presented. The method is based on analysis of transverse resonance vibration of a cantilevered tungsten micro-needle (1,5 divided by 2 mm long, 30 divided by 40 mkm in diameter) having the specimen stuck on its free sharp end. The method is accurate to within 0.1% for specimens with masses 0.1 divided by 0.01 mg. Absorption isotherms for water uptake by triclinic (P1), monoclinic (P2(1) ) and tetragonal (P4(3)2(1)2) crystals as well as by amorphous films of hen egg-white lysozyme are obtained. Hydration of lysozyme molecule is shown to be highly dependent on molecular packing in the sample both at low and high relative humidities.

  8. Specific Ion Effects: Why the Properties of Lysozyme in Salt Solutions Follow a Hofmeister Series

    PubMed Central

    Boström, M.; Williams, D. R. M.; Ninham, B. W.

    2003-01-01

    Protein solubility in aqueous solutions depends in a complicated and not well understood way on pH, salt type, and salt concentration. Why for instance does the use of two different monovalent salts, potassium thiocyanate and potassium chloride, produce such different results? One important and previously neglected source of ion specificity is the ionic dispersion potential that acts between each ion and the protein. This attractive potential is found to be much stronger for SCN− than it is for Cl−. We present model calculations, performed within a modified ion-specific double-layer theory, that demonstrate the large effect of including these ionic dispersion potentials. The results are consistent with experiments performed on hen egg-white lysozymes and on neutral black lipid membranes. The calculated surface pH and net lysozyme charge depend strongly on the choice of anion. We demonstrate that the lysozyme net charge is larger, and the corresponding Debye length shorter, in a thiocyanate salt solution than in a chloride salt solution. Recent experiments have suggested that pKa values of histidines depend on salt concentration and on ionic species. We finally demonstrate that once ionic dispersion potentials are included in the theory these results can quantitatively be reinterpreted in terms of a highly specific surface pH (and a salt-independent pKa). PMID:12885620

  9. The Averaged Face Growth Rates of lysozyme Crystals: The Effect of Temperature

    NASA Technical Reports Server (NTRS)

    Nadarajah, Arunan; Forsythe, Elizabeth L.; Pusey, Marc L.

    1995-01-01

    Measurements of the averaged or macroscopic face growth rates of lysozyme crystals are reported here for the (110) face of tetragonal lysozyme, at three sets of pH and salt concentrations, with temperatures over a 4-22 C range for several protein concentrations. The growth rate trends with supersaturation were similar to previous microscopic growth rate measurements. However, it was found that at high super-saturations the growth rates attain a maximum and then start decreasing. No 'dead zone' was observed but the growth rates were found to approach zero asymptotically at very low super-saturations. The growth rate data also displayed a dependence on pH and salt concentration which could not be characterized solely by the super-saturation. A complete mechanism for lysozyme crystal growth, involving the formation of an aggregate growth unit, mass transport of the growth unit to the crystal interface and faceted crystal growth by growth unit addition, is suggested. Such a mechanism may provide a more consistent explanation for the observed growth rate trends than those suggested by other investigators. The nutrient solution interactions leading to the formation of the aggregate growth unit may, thus, be as important as those occurring at the crystal interface and may account for the differences between small molecule and protein crystal growth.

  10. Comparison of two codon optimization strategies enhancing recombinant Sus scrofa lysozyme production in Pichia pastoris.

    PubMed

    Zhu, D; Cai, G; Wu, D; Lu, J

    2015-05-16

    Lysozyme has played an important role in animal feed additive industry, food additive industry and biological engineering. For improving expression efficiency of recombinant lysozyme from Sus scrofa, two genes respectively designed by the most used codon optimization strategies, "one amino acid one codon" and "codon randomization", were synthesized and expressed in Pichia pastoris X—33. At shaking flask level, Sus scrofa lysozyme (SSL) under two conditions had a highest activity of 153.33±10.41 and 538.33±15.18 U/mL after a 5 days induction of 1% methanol, with secreted protein concentration 80.03±1.94 and 239.60±4.16 mg/L, respectively. Compared with the original SSL gene, the expression of optimized SSL gene by the second strategy showed a 2.6 fold higher level, while the first method had no obvious improvement in production. In total secreted protein, the proportions of recombinant SSL encoded by the original gene, first method optimized gene and the second—strategy optimized one were 75.06±0.25%, 74.56±0.14% and 79.00±0.14%, respectively, with the same molecular weight about 18 kDa, optimum acidity pH 6.0 and optimum temperature 35degC.

  11. Lipids, proteins, phenolic composition, antioxidant and antibacterial activities of seeds of peanuts (Arachis hypogaea l) cultivated in Tunisia.

    PubMed

    Sebei, Khaled; Gnouma, Asma; Herchi, Wahid; Sakouhi, Faouzi; Boukhchina, Sadok

    2013-01-01

    Fatty acid composition of peanut seed oil in four varieties cultivated in Tunisia showed that linoleic (C18:2), oleic (C18:1) and palmitic (C16) acids account for more than 84% for Chounfakhi and Massriya and for more than 85% of the total fatty acids of Trabilsia and Sinya seed oil respectively. Seed oil contents were significantly different (P ≤ 0.05) and did not exceed 48%. The study of total phenolics revealed that Chounfakhi contained more total phenolics (2.1 mg GAE/g DW), followed by the Massriya and Sinya cultivars (1.35 mg GAE/g DW for each); Trabilsia presented the lowest total phenolic content with 1 mg GAE/g DW. Considerable antiradical ability was found, especially in the Trabilsia peanut seed cultivar (IC50 = 1550 μg/ml), the Massriya and Sinya cultivars had, respectively, 720 and 820 mg/ml IC50. In the Massriya variety the sterol fraction showed antibacterial activity against Listeria ivanovii, Listeria inocua, Pseudomonas aeruginosa, Staphylococus aureus, Enterococcus hirae and Bacillus cereus.

  12. The role of ficolin-like protein (PcFLP1) in the antibacterial immunity of red swamp crayfish (Procambarus clarkii).

    PubMed

    Dai, Yun-Jia; Wang, Yu-Qing; Zhang, Ying-Hao; Liu, Yan; Li, Jin-Quan; Wei, Shun; Zhao, Li-Juan; Zhou, Yong-Can; Lin, Li; Lan, Jiang-Feng

    2017-01-01

    In invertebrates, ficolin-like proteins (FLPs) play important roles in innate immunity against pathogens. Previous studies primarily investigated the functions of FLPs in immune recognition, activation, and regulation. However, limited research has examined the functions of FLPs as immune effectors. In this work, a ficolin-like protein was identified in red swam crayfish (Procambarus clarkii) and designated as PcFLP1. Quantitative RT-PCR and western blot were employed to analyze the distribution and expression profiles of PcFLP1 in the tissues of the crayfish. The results indicated that PcFLP1 was present in all tested tissues, including hemocytes, heart, hepatopancreas, gill, stomach, and mid-intestine. The expression level of PcFLP1 was up-regulated in hemocytes, hepatopancreas and mid-intestines of the crayfish challenged with Vibrio parahaemolyticus. Further study demonstrated that PcFLP1 could protect the hepatopancreatic cells of crayfish from V. parahaemolyticus infection. The recombinant PcFLP1 enhanced bacterial elimination in crayfish, whereas the antibacterial action was inhibited after PcFLP1 was knocked down. Furthermore, PcFLP1 could bound to bacteria and inhibited bacterial replication. These results demonstrated that PcFLP1 plays an important role in the anti-Vibrio immunity of red swamp crayfish.

  13. Experimental study of albumin and lysozyme adsorption onto acrylic acid (AA) and 2-hydroxyethyl methacrylate (HEMA) surfaces.

    PubMed

    Moradi, Omid; Modarress, Hamid; Noroozi, Mehdi

    2004-03-01

    Many commercial soft contact lenses are based on poly-2-hydroxyethyl methacrylate (HEMA) and acrylic acid (AA) hydrogels. The adsorption of proteins, albumin and lysozyme, on such contact lens surfaces may cause problems in their applications. In this work the adsorption of proteins, albumin and lysozyme, on hydrogel surfaces, AA and HEMA, was investigated as a function of concentration of protein. Also the effects of pH and ionic strength of protein solution on the adsorption of protein were examined. The obtained results indicated that the degree of adsorption of protein increased with the concentration of protein, and the adsorption of albumin on HEMA surface at the studied pHs (6.2-8.6) was higher than AA surface, whereas the adsorption of lysozyme on AA surface at the same pHs was higher than HEMA. The change in ionic strength of protein solution affected the proteins adsorption on both AA and HEMA surfaces. Also, the amount of sodium ions deposited on the AA surface was much higher than HEMA surface. This effect can be related to the negative surface charge of AA and its higher tendency for adsorption of sodium ions compared to the HEMA surface.

  14. Effects of mercuric chloride on chemiluminescent response of phagocytes and tissue lysozyme activity in Tilapia, Oreochromis aureus

    SciTech Connect

    Low, K.W.; Sin, Y.M.

    1995-02-01

    Phagocytosis is an important defense mechanism against foreign pathogenic organisms. The cells involved are phagocytes which are comprised of peripheral blood monocytes (tissue macrophages) and polymorphonuclear (PMN) leucocytes. These cells can be activated by either particulate or soluble stimuli and undergo a respiratory burst from which several reactive oxygen species (ROS) can be formed. The reactive oxygen species and some hydrolases generated in the cells are the major antibacterial agents released during phagocytosis. Chemiluminescence (CL) is emitted, in vitro, from phagocytizing human PMN neutrophils. A similar CL response was also encountered in fish phagocytes. ROS was the causative agent of the CL emitted during in vitro phagocytosis. Phagocytic activity can be monitored by measuring the CL response of the phagocytes. Lysozyme is one of the potent hydrolases which are involved in the destruction of pathogens during phagocytosis. In fish, it was found predominantly in haematopoietic tissues, PMN leucocytes and moncytes. This enzyme has been shown to have antibacterial activity against several pathogens in fish. A combined oxidative and hydrolytic attack upon the engulfed pathogens allow phagocytes to kill infectious agents effectively. However, severe suppression or enhancement of these two functions caused by some exogenous factors may be detrimental to the host tissues. It has been reported that inorganic mercury could inhibit, in vitro, the respiratory burst and the microbicidal activities of human PMN leucocytes. It was also reported that increased in vitro release of lysozyme was found in mercury-treated human PMN leucocytes. However, such work has not been reported in fish. The aim of this research was to examine whether mercury could exert similar effects on the CL response in phagocytes and tissue lysozyme activity in fish after they were exposed to different concentrations of mercuric chloride over a period of 3 wks. 17 refs., 1 fig., 1 tab.

  15. [Purification and characterization of a lysozyme from a marine microorganism].

    PubMed

    Zou, Yan-Li; Sun, Mi; Wang, Yue-Jun

    2005-05-01

    A novel lysozyme was purified from a marine microorganism and its major characteristics were studied. Cell-free supernatant was prepared by centrifugation of culture broth, ultrafiltration using a hollow fiber (molecular weight cut off, 50kD) and concentration using a hollow fiber (molecular weight cut off, 10kD). The crude lysozyme was purified 34.7 fold to electrophoretic homogeneity with a recovery of 24.1% by CM-Sepharose FF cationic-exchange and Sephadex G-100 gel chromatography. The relative molecular weight of this lysozyme was determined as about 39 kD. The optimum pH and temperature towards Micrococcus lysodleikticus were pH 8.0 and 35 degrees C respectively, and the enzyme was stable at temperature below 50 degrees C and pH 5.0 - 10.0. The lysozyme activity was slightly enhanced by Zn2+ and Cu2+ and slightly inhibited by Mn2+ and Ag+. The lysozyme showed good compatibility to many common chemical agents such as EDTA (0.1%) and KH2 PO4 (1.0%). The lysozyme had broad-spectrum against many bacteria, including a number of pathogens, which were resistant to egg-white lysozyme.

  16. The Effects of Acetate Buffer Concentration on Lysozyme Solubility

    NASA Technical Reports Server (NTRS)

    Forsythe, Elizabeth L.; Pusey, Marc L.

    1996-01-01

    The micro-solubility column technique was employed to systematically investigate the effects of buffer concentration on tetragonal lysozyme solubility. While keeping the NaCl concentrations constant at 2%, 3%, 4%, 5% and 7%, and the pH at 4.0, we have studied the solubility of tetragonal lysozyme over an acetate buffer concentration range of 0.01M to 0.5M as a function of temperature. The lysozyme solubility decreased with increasing acetate concentration from 0.01M to 0.1M. This decrease may simply be due to the net increase in solvent ionic strength. Increasing the acetate concentration beyond 0.1M resulted in an increase in the lysozyme solubility, which reached a peak at - 0.3M acetate concentration. This increase was believed to be due to the increased binding of acetate to the anionic binding sites of lysozyme, preventing their occupation by chloride. In keeping with the previously observed reversal of the Hoffmeister series for effectiveness of anions in crystallizing lysozyme, acetate would be a less effective precipitant than chloride. Further increasing the acetate concentration beyond 0.3M resulted in a subsequent gradual decrease in the lysozyme solubility at all NaCl concentrations.

  17. Separation of lysozyme using superparamagnetic carboxymethyl chitosan nanoparticles.

    PubMed

    Sun, Jun; Su, Yujie; Rao, Shengqi; Yang, Yanjun

    2011-08-01

    Functionalized Fe(3)O(4) nanoparticles conjugated with polyethylene glycol (PEG) and carboxymethyl chitosan (CM-CTS) were developed and used as a novel magnetic absorbing carrier for the separation and purification of lysozyme from the aqueous solution and chicken egg white, respectively. The morphology of magnetic CM-CTS nanoparticles was observed by transmission electron microscope (TEM). It was found that the diameter of superparamagnetic carboxymethyl chitosan nanoparticles (Fe(3)O(4) (PEG+CM-CTS)) was about 15 nm, and could easily aggregate by a magnet when suspending in the aqueous solution. The adsorption capacity of lysozyme onto the superparamagnetic Fe(3)O(4) (PEG+CM-CTS) nanoparticles was determined by changing the medium pH, temperature, ionic strength and the concentration of lysozyme. The maximum adsorption loading reached 256.4 mg/g. Due to the small diameter, the adsorption equilibrium of lysozyme onto the nanoparticles reached very quickly within 20 min. The adsorption equilibrium of lysozyme onto the superparamagnetic nanoparticles fitted well with the Langmuir model. The nanoparticles were stable when subjected to six repeated adsorption-elution cycles. Separation and purification were monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The lysozyme was purified from chicken egg white in a single step had higher purity, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Considering that the superparamagnetic nanoparticles possess the advantages of high efficiency, cost-effectiveness and excellent binding of a larger amount of lysozyme and easier separation from the reaction system, thus this type of superparamagnetic nanoparticles would bring advantages to the conventional separation techniques of lysozyme from chicken egg white.

  18. The effect of protein–precipitant interfaces and applied shear on the nucleation and growth of lysozyme crystals

    SciTech Connect

    Reis, Nuno M.; Chirgadze, Dimitri Y.; Blundell, Tom L.; Mackley, Malcolm R.

    2009-11-01

    The nucleation of lysozyme in microbatch experiments was linked to the formation of protein–precipitant interfaces. The use of oscillatory shear allowed decreasing the nucleation rate and extending the growth period for lysozyme crystals, presumably through the control of the number of interfaces and removal of impurities or defects. This paper is concerned with the effect of protein–precipitant interfaces and externally applied shear on the nucleation and growth kinetics of hen egg-white lysozyme crystals. The early stages of microbatch crystallization of lysozyme were explored using both optical and confocal fluorescence microscopy imaging. Initially, an antisolvent (precipitant) was added to a protein drop and the optical development of the protein–precipitant interface was followed with time. In the presence of the water-soluble polymer poly(ethylene glycol) (PEG) a sharp interface was observed to form immediately within the drop, giving an initial clear separation between the lighter protein solution and the heavier precipitant. This interface subsequently became unstable and quickly developed within a few seconds into several unstable ‘fingers’ that represented regions of high concentration-gradient interfaces. Confocal microscopy demonstrated that the subsequent nucleation of protein crystals occurred preferentially in the region of these interfaces. Additional experiments using an optical shearing system demonstrated that oscillatory shear significantly decreased nucleation rates whilst extending the growth period of the lysozyme crystals. The experimental observations relating to both nucleation and growth have relevance in developing efficient and reliable protocols for general crystallization procedures and the controlled crystallization of single large high-quality protein crystals for use in X-ray crystallography.

  19. Sequence-motif Detection of NAD(P)-binding Proteins: Discovery of a Unique Antibacterial Drug Target

    NASA Astrophysics Data System (ADS)

    Hua, Yun Hao; Wu, Chih Yuan; Sargsyan, Karen; Lim, Carmay

    2014-09-01

    Many enzymes use nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate (NAD(P)) as essential coenzymes. These enzymes often do not share significant sequence identity and cannot be easily detected by sequence homology. Previously, we determined all distinct locally conserved pyrophosphate-binding structures (3d motifs) from NAD(P)-bound protein structures, from which 1d sequence motifs were derived. Here, we aim to establish the precision of these 3d and 1d motifs to annotate NAD(P)-binding proteins. We show that the pyrophosphate-binding 3d motifs are characteristic of NAD(P)-binding proteins, as they are rarely found in nonNAD(P)-binding proteins. Furthermore, several 1d motifs could distinguish between proteins that bind only NAD and those that bind only NADP. They could also distinguish between NAD(P)-binding proteins from nonNAD(P)-binding ones. Interestingly, one of the pyrophosphate-binding 3d and corresponding 1d motifs was found only in enoyl-acyl carrier protein reductases, which are enzymes essential for bacterial fatty acid biosynthesis. This unique 3d motif serves as an attractive novel drug target, as it is conserved across many bacterial species and is not found in human proteins.

  20. Ocatin. A Novel Tuber Storage Protein from the Andean Tuber Crop Oca with Antibacterial and Antifungal Activities1

    PubMed Central

    Flores, Teresita; Alape-Girón, Alberto; Flores-Díaz, Marietta; Flores, Hector E.

    2002-01-01

    The most abundant soluble tuber protein from the Andean crop oca (Oxalis tuberosa Mol.), named ocatin, has been purified and characterized. Ocatin accounts for 40% to 60% of the total soluble oca tuber proteins, has an apparent molecular mass of 18 kD and an isoelectric point of 4.8. This protein appears to be found only in tubers and is accumulated only within the cells of the pith and peridermis layers (peel) of the tuber as it develops. Ocatin inhibits the growth of several phytopathogenic bacteria (Agrobacterium tumefaciens, Agrobacterium radiobacter, Serratia marcescens, and Pseudomonas aureofaciens) and fungi (Phytophthora cinnamomi, Fusarium oxysporum, Rhizoctonia solani, and Nectria hematococcus). Ocatin displays substantial amino acid sequence similarity with a widely distributed group of intracellular pathogenesis-related proteins with a hitherto unknown biological function. Our results showed that ocatin serves as a storage protein, has antimicrobial properties, and belongs to the Betv 1/PR-10/MLP protein family. Our findings suggest that an ancient scaffolding protein was recruited in the oca tuber to serve a storage function and that proteins from the Betv 1/PR-10/MLP family might play a role in natural resistance to pathogens. PMID:11950978

  1. Antibacterial effect of ultrafine nanodiamond against gram-negative bacteria Escherichia coli

    NASA Astrophysics Data System (ADS)

    Chatterjee, Anindita; Perevedentseva, Elena; Jani, Mona; Cheng, Chih-Yuan; Ye, Ying-Siou; Chung, Pei-Hua; Cheng, Chia-Liang

    2015-05-01

    We investigate the antibacterial effect of ultrafine nanodiamond particles with an average size of 5 nm against the gram-negative bacteria Escherichia coli (E. coli). UV-visible, Raman spectroscopy, and scanning electron microscopy (SEM) have been employed to elucidate the nature of the interaction. The influence on bacterial growth was monitored by measuring optical densities of E. coli at 600 nm as a function of time in the presence of carboxylated nanodiamond (cND) particles (100 μg/ml) in highly nutritious liquid Luria-Bertani medium. The SEM images prove that cND particles are attached to the bacterial cell wall surface and some portion of the bacterial cell wall undergoes destruction. Due to the change of the protein structure on the bacterial wall, a small Raman shift in the region of 1400 to 1700 cm-1 was observed when E. coli interacted with cNDs. Raman mapping images show strong evidence of cND attachment at the bacterial cell wall surface. Electrotransformation of E. coli with a fluorescent protein markers experiment demonstrated that the interaction mechanisms are different for E. coli treated with cND particles, E. coli by lysozyme treatment, and E. coli that suffer lysis.

  2. Antibacterial effect of ultrafine nanodiamond against gram-negative bacteria Escherichia coli.

    PubMed

    Chatterjee, Anindita; Perevedentseva, Elena; Jani, Mona; Cheng, Chih-Yuan; Ye, Ying-Siou; Chung, Pei-Hua; Cheng, Chia-Liang

    2015-05-01

    We investigate the antibacterial effect of ultrafine nanodiamond particles with an average size of 5 nm against the gram-negative bacteria Escherichia coli (E. coli). UV-visible, Raman spectroscopy, and scanning electron microscopy (SEM) have been employed to elucidate the nature of the interaction. The influence on bacterial growth was monitored by measuring optical densities of E. coli at 600 nm as a function of time in the presence of carboxylated nanodiamond (cND) particles (100 μg/ml ) in highly nutritious liquid Luria-Bertani medium. The SEM images prove that cND particles are attached to the bacterial cell wall surface and some portion of the bacterial cell wall undergoes destruction. Due to the change of the protein structure on the bacterial wall, a small Raman shift in the region of 1400 to 1700 cm⁻¹ was observed when E. coli interacted with cNDs. Raman mapping images show strong evidence of cND attachment at the bacterial cell wall surface. Electrotransformation of E. coli with a fluorescent protein markers experiment demonstrated that the interaction mechanisms are different for E. coli treated with cND particles, E. coli by lysozyme treatment, and E. coli that suffer lysis.

  3. Liquid-liquid phase separation in supersaturated lysozyme solutions and associated precipitate formation/crystallization

    NASA Astrophysics Data System (ADS)

    Muschol, Martin; Rosenberger, Franz

    1997-08-01

    Using cloud point determinations, the phase boundaries (binodals) for metastable liquid-liquid (L-L) separation in supersaturated hen egg white lysozyme solutions with 3%, 5%, and 7% (w/v) NaCl at pH=4.5 and protein concentrations c between 40 and 400 mg/ml were determined. The critical temperature for the binodal increased approximately linearly with salt concentration. The coexisting liquid phases both remained supersaturated but differed widely in protein concentration. No salt repartitioning was observed between the initial and the two separated liquid phases. After the L-L separation, due to the presence of the high protein concentration phase, crystallization occurred much more rapidly than in the initial solution. At high initial protein concentrations, a metastable gel phase formed at temperatures above the liquid binodal. Both crystal nucleation and gel formation were accelerated in samples that had been cycled through the binodal. Solutions in the gel and L-L regions yielded various types of precipitates. Based on theoretical considerations, previous observations with other proteins, and our experimental results with lysozyme, a generic phase diagram for globular proteins is put forth. A limited region in the (T,c) plane favorable for the growth of protein single crystals is delineated.

  4. Liquid-Liquid Phase Separation in Supersaturated Lysozyme Solutions and Associated Precipitate Formation/Crystallization

    NASA Technical Reports Server (NTRS)

    Muschol, Martin; Rosenberger, Franz

    1997-01-01

    Using cloud point determinations, the phase boundaries (binodals) for metastable liquid-liquid (L-L) separation in supersaturated hen egg white lysozyme solutions with 3%, 5%, and 7% (wlv) NaCl at pH= 4.5 and protein concentrations c between 40 and 400 mg/ml were determined. The critical temperature for the binodal increased approximately linearly with salt concentration. The coexisting liquid phases both remained supersaturated but differed widely in protein concentration. No salt repartitioning was observed between the initial and the two separated liquid phases. After the L-L separation, due to the presence of the high protein concentration phase, crystallization occurred much more rapidly than in the initial solution. At high initial protein concentrations, a metastable gel phase formed at temperatures above the liquid binodal. Both crystal nucleation and gel formation were accelerated in samples that had been cycled through the binodal. Solutions in the gel and L-L regions yielded various types of precipitates. Based on theoretical considerations, previous observations with other proteins, and our experimental results with lysozyme, a generic phase diagram for globular proteins is put forth. A limited region in the (T,c) plane favorable for the growth of protein single crystals is delineated.

  5. Refolding of denatured lysozyme by water-in-oil microemulsions of sucrose fatty acid esters.

    PubMed

    Noritomi, Hidetaka; Takasugi, Tsubasa; Kato, Satoru

    2008-04-01

    Water-in-oil (w/o) microemulsion of sucrose fatty acid ester was used to renature denatured hen egg white lysozyme without aggregation. After lysozyme was denatured in 5 M guanidine hydrochloride for 24 h, the resultant denatured lysozyme was held in the microemulsion, overnight at 25 degrees C. Renatured lysozyme was transferred from the microemulsion phase to the recovery aqueous phase by conventional liquid-liquid extraction. The enzymatic activity of the recovered lysozyme was 93%.

  6. Investigation on the interaction of cefpirome sulfate with lysozyme by fluorescence quenching spectroscopy and synchronous fluorescence spectroscopy.

    PubMed

    Han, Rong; Liu, Baosheng; Li, Gaixia; Zhang, Qiuju

    2016-03-01

    The reaction mechanism of cefpirome sulfate with lysozyme at different temperatures (298, 310 and 318 K) was investigated using fluorescence quenching and synchronous fluorescence spectroscopy under simulated physiological conditions. The results clearly demonstrated that cefpirome sulfate caused strong quenching of the fluorescence of lysozyme by a static quenching mechanism. The binding constants obtained using the above methods were of the same order of magnitude and very similar. Static electric forces played a key role in the interaction between cefpirome sulfate and lysozyme, and the number of binding sites in the interaction was close to 1. The values of Hill's coefficients were > 1, indicating that drugs or proteins showed a very weakly positive cooperativity in the system. In addition, the conclusions obtained from the two methods using the same equation were consistent. The results indicated that synchronous fluorescence spectrometry could be used to study the binding mechanism between drug and protein, and was a useful supplement to the fluorescence quenching method. In addition, the effect of cefpirome sulfate on the secondary structure of lysozyme was analyzed using circular dichroism spectroscopy.

  7. Partial Restoration of Antibacterial Activity of the Protein Encoded by a Cryptic Open Reading Frame (cyt1Ca) from Bacillus thuringiensis subsp. israelensis by Site-Directed Mutagenesis

    PubMed Central

    Itsko, Mark; Manasherob, Robert; Zaritsky, Arieh

    2005-01-01

    Insecticidal crystal proteins of Bacillus thuringiensis belong to two unrelated toxin families: receptor-specific Cry toxins against insects and Cyt toxins that lyse a broad range of cells, including bacteria, via direct binding to phospholipids. A new cyt-like open reading frame (cyt1Ca) encoding a 60-kDa protein, has recently been discovered (C. Berry et al., Appl. Environ. Microbiol. 68:5082-5095, 2002). Cyt1Ca displays the structure of a two-domain fusion protein: the N-terminal moiety resembles the full-length Cyt toxins, and the C-terminal moiety is similar to the receptor-binding domains of several ricin-like toxins, such as Mtx1. Neither the larvicidal activity of cyt1Ca expressed in Escherichia coli nor the hemolytic effect of His-tagged purified Cyt1Ca has been observed (R. Manasherob et al., unpublished). This was attributed to five amino acid differences between the sequences of its N-terminal moiety and Cyt1Aa. The 3′ end of cyt1Ca was truncated (removing the ricin-binding domain of Cyt1Ca), and six single bases were appropriately changed by site-directed mutagenesis, sequentially replacing the noncharged amino acids by charged ones, according to Cyt1Aa, to form several versions. Expression of these mutated cyt1Ca versions caused loss of the colony-forming ability of the corresponding E. coli cells to different extents compared with the original gene. In some mutants this antibacterial effect was associated by significant distortion of cell morphology and in others by generation of multiple inclusion bodies spread along the cell envelope. The described deleterious effects of mutated cyt1Ca versions against E. coli may reflect an evolutionary relationship between Cyt1Aa and Cyt1Ca. PMID:16159771

  8. Nucleation and convection effects in protein crystal growth

    NASA Technical Reports Server (NTRS)

    Rosenberger, Franz (Principal Investigator)

    1996-01-01

    The following activities are reported on: repartitioning of NaCl and protein impurities in lysozyme crystallization; dependence of lysozyme growth kinetics on step sources and impurities; facet morphology response to nonuniformities in nutrient and impurity supply; interactions in undersaturated and supersaturated lysozyme solutions; heterogeneity determination and purification of commercial hen egg white lysozyme; nonlinear response of layer growth dynamics in the mixed kinetics-bulk transport regime; development of a simultaneous multiangle light scattering technique; and x-ray topography of tetragonal lysozyme grown by the temperature-control technique.

  9. Lysozyme and defense peptides as suppressors of phenoloxidase activity in Galleria mellonella.

    PubMed

    Zdybicka-Barabas, Agnieszka; Mak, Paweł; Jakubowicz, Teresa; Cytryńska, Małgorzata

    2014-09-01

    The prophenoloxidase (proPO) cascade supplies quinones and other reactive compounds for melanin formation, protein cross-linking, hemolymph coagulation, and killing of microbial invaders as well as parasites. The high cytotoxicity of the generated compounds requires a strict control of the activation of the proPO system and phenoloxidase (PO) activity to minimize damage to host tissues and cells. The PO activity in hemolymph of Escherichia coli challenged Galleria mellonella larvae increased, with a temporal drop 1 h after the challenge, reaching the highest level 24 h after the challenge. In the present study, a potential role of G. mellonella defense peptides and lysozyme in controlling the proPO system was investigated. The effects of purified defense peptides (anionic peptides 1 and 2, cecropin D-like peptide, Galleria defensin, proline-rich peptides 1 and 2) and lysozyme were analyzed. Four compounds, namely lysozyme, Galleria defensin, proline-rich peptide 1, and anionic peptide 2, decreased the hemolymph PO activity considerably, whereas the others did not affect the enzyme activity level. Our results indicate that these hemolymph factors could play multiple and distinct roles in the insect immune response.

  10. Interactions between high pressure homogenization and antimicrobial activity of lysozyme and lactoperoxidase.

    PubMed

    Vannini, L; Lanciotti, R; Baldi, D; Guerzoni, M E

    2004-07-15

    It was the objective of this work to evaluate the effect of high pressure homogenization on the activity of antimicrobial enzymes such as lysozyme and lactoperoxidase against a selected group of Gram positive and Gram negative species inoculated in skim milk. Lactobacillus helveticus, Lactobacillus plantarum and Listeria monocytogenes were the most pressure resistant species while Bacillus subtilis, Pseudomonas putida, Salmonella typhimurium, Staphylococcus aureus, Proteus vulgaris and Salmonella enteritidis were found to be very sensitive to the hyperbaric treatment. The enzyme addition enhanced the instantaneous pressure efficacy on almost all the considered species as indicated by their instantaneous viability loss following the treatment. Moreover, the combination of the enzyme and high pressure homogenization significantly affected the recovery and growth dynamics of several of the considered species. Although L. monocytogenes was slightly sensitive to pressure, the combination of the two stress factors induced a significant viability loss within 3 h and an extension of lag phases in skim milk during incubation at 37 degrees C. The hypothesis formulated in this work is that the interaction of high pressure homogenization and lysozyme or lactoperoxidase is associated to conformational modifications of the two proteins with a consequent enhancement of their activity. This hypothesis is supported by the experimental results also regarding the increased antimicrobial activity against L. plantarum of the previously pressurised lysozyme with respect to that of the native enzyme.

  11. Lysozyme complexes with thermo- and pH-responsive PNIPAM- b-PAA block copolymer

    NASA Astrophysics Data System (ADS)

    Pippa, Natassa; Meristoudi, Anastasia; Pispas, Stergios; Demetzos, Costas

    2017-02-01

    Lysozyme is an enzyme responsible for the damage of bacterial cell walls and is abundant in a number of secretions such as tears and human milk. In the present study, we investigated the structure, the physicochemical characteristics, and the temperature-responsiveness of lysozyme complexes with poly( N-isopropylacrylamide)- b-poly(acrylic acid) block polyelectrolyte in aqueous media. A gamut of light-scattering techniques and fluorescence spectroscopy were used in order to examine the complexation process, as well as the structure, solution behavior, and temperature response of the nanosized complexes. The concentration of copolymer polyelectrolyte was kept constant. The values of the scattering intensity, I 90, which is proportional to the mass of the species in solution, increased gradually as a function of C LYS, providing proof of the occurring complexation, while the size of the nanostructures decreased. The structure of the complexes became more open as the C LYS increased. The increase of the salinity did not affect the structural characteristics of the supramolecular nanoparticulate aggregates. On the other hand, the physicochemical and structural characteristics of the complexes changed upon increasing temperature, and the changes depended on the initial ratio block polyelectrolyte/lysozyme. The knowledge on developing block polyelectrolyte/protein complexes through electrostatic interactions, obtained from this investigation, may be applied to the design of nutraceuticals.

  12. Structural Relationships in the Lysozyme Superfamily: Significant Evidence for Glycoside Hydrolase Signature Motifs

    PubMed Central

    Wohlkönig, Alexandre; Huet, Joëlle; Looze, Yvan; Wintjens, René

    2010-01-01

    Background Chitin is a polysaccharide that forms the hard, outer shell of arthropods and the cell walls of fungi and some algae. Peptidoglycan is a polymer of sugars and amino acids constituting the cell walls of most bacteria. Enzymes that are able to hydrolyze these cell membrane polymers generally play important roles for protecting plants and animals against infection with insects and pathogens. A particular group of such glycoside hydrolase enzymes share some common features in their three-dimensional structure and in their molecular mechanism, forming the lysozyme superfamily. Results Besides having a similar fold, all known catalytic domains of glycoside hydrolase proteins of lysozyme superfamily (families and subfamilies GH19, GH22, GH23, GH24 and GH46) share in common two structural elements: the central helix of the all-α domain, which invariably contains the catalytic glutamate residue acting as general-acid catalyst, and a β-hairpin pointed towards the substrate binding cleft. The invariant β-hairpin structure is interestingly found to display the highest amino acid conservation in aligned sequences of a given family, thereby allowing to define signature motifs for each GH family. Most of such signature motifs are found to have promising performances for searching sequence databases. Our structural analysis further indicates that the GH motifs participate in enzymatic catalysis essentially by containing the catalytic water positioning residue of inverting mechanism. Conclusions The seven families and subfamilies of the lysozyme superfamily all have in common a β-hairpin structure which displays a family-specific sequence motif. These GH β-hairpin motifs contain potentially important residues for the catalytic activity, thereby suggesting the participation of the GH motif to catalysis and also revealing a common catalytic scheme utilized by enzymes of the lysozyme superfamily. PMID:21085702

  13. Immobilization of lysozyme on polyvinylalcohol films for active packaging applications.

    PubMed

    Conte, A; Buonocore, G G; Bevilacqua, A; Sinigaglia, M; Del Nobile, M A

    2006-04-01

    A new technique for the immobilization of lysozyme onto the surface of polyvinylalcohol films is presented. The active compound was sprayed along with a suitable bonding agent onto the surface of the cross-linked polymeric matrix. Active compound release tests determined the amount of lysozyme immobilized on the film surface. With the use of Micrococcus lysodeikticus, the antimicrobial activity of the films was determined and the results correlated with the amount of immobilized lysozyme. This new technique was effective for immobilizing the enzyme, and the developed films were active against the test microorganism. Results were compared with those obtained with a different immobilizing technique, in which the active compound was bound into the bulk of the polymeric film. As expected, the surface-immobilized lysozyme films have a higher antimicrobial activity than bulk-bound films.

  14. Dielectric and gravimetric studies of water binding to lysozyme.

    PubMed

    Bone, S

    1996-08-01

    Time domain dielectric spectroscopy and hydration isotherm measurements as a function of temperature have been applied to hydrated lysozyme powder. Two dielectric dispersions were identified, the first centred at approximately 8 MHz and a second above 1 GHz. The higher dispersion is considered to be the result of rotational relaxation of water molecules bound to the enzyme. In this case the results indicate the existence of a population of 32 water molecules per lysozyme molecule which are irrotationally bound to the lysozyme structure. A larger population of water molecules is relatively free to respond to the electric field and exhibits a dipole moment close to that of vapour phase water molecules. Multi-temperature hydration isotherm measurements are used to calculate enthalpies and entropies associated with the binding of water to lysozyme. Discontinuities both in dielectric and in thermodynamic characteristics in the range 10-14% hydration are interpreted as a re-ordering of the water structure on the enzyme surface.

  15. 21 CFR 862.1490 - Lysozyme (muramidase) test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... serum, plasma, leukocytes, and urine. Lysozyme measurements are used in the diagnosis and treatment of monocytic leukemia and kidney disease. (b) Classification. Class I (general controls). The device is...

  16. Bioengineered lysozyme in combination therapies for Pseudomonas aeruginosa lung infections

    PubMed Central

    Griswold, Karl E; Bement, Jenna L; Teneback, Charlotte C; Scanlon, Thomas C; Wargo, Matthew J; Leclair, Laurie W

    2014-01-01

    There is increasing urgency in the battle against drug-resistant bacterial pathogens, and this public health crisis has created a desperate need for novel antimicrobial agents. Recombinant human lysozyme represents one interesting candidate for treating pulmonary infections, but the wild type enzyme is subject to electrostatic mediated inhibition by anionic biopolymers that accumulate in the infected lung. We have redesigned lysozyme’s electrostatic potential field, creating a genetically engineered variant that is less susceptible to polyanion inhibition, yet retains potent bactericidal activity. A recent publication demonstrated that the engineered enzyme outperforms wild type lysozyme in a murine model of Pseudomonas aeruginosa lung infection. Here, we expand upon our initial studies and consider dual therapies that combine lysozymes with an antimicrobial peptide. Consistent with our earlier results, the charge modified lysozyme combination outperformed its wild type counterpart, yielding more than an order-of-magnitude reduction in bacterial burden following treatment with a single dose. PMID:24637705

  17. Preliminary crystallographic examination of a novel fungal lysozyme from Chalaropsis

    NASA Technical Reports Server (NTRS)

    Carter, Daniel C.; He, Xiao-Min; Lyne, James E.; Stubbs, Gerald; Hash, John H.

    1990-01-01

    The lysozyme from the fungus of the Chalaropsis species has been crystallized. This lysozyme displays no sequence homology with avian, phage, or mammalian lysozymes, however, preliminary studies indicate significant sequence homology with the bacterial lysozyme from Streptomyces. Both enzymes are unusual in possessing beta-1,4-N-acetylmuramidase and beta-1,4-N,6-O-diacetylmuramidase activity. The crystals grow from solutions of ammonium sulfate during growth periods from several months to a year. The space group is P2(1)2(1)2(1) with a = 34.0 A, b = 42.6 A, c = 122.1 A. Preliminary data indicate that there is 1 molecule/asymmetric unit.

  18. Relationship between β-relaxation and structural stability of lysozyme: Microscopic insight on thermostabilization mechanism by trehalose from Raman spectroscopy experiments

    SciTech Connect

    Hédoux, Alain Paccou, Laurent; Guinet, Yannick

    2014-06-14

    Raman investigations were carried out in the low-frequency and amide I regions on lysozyme aqueous solutions in absence and presence of trehalose. Raman spectroscopy gives the unique opportunity to analyze the protein and solvent dynamics in the low-frequency range while monitoring the unfolding process by capturing the spectrum of the amide I band. From the analysis of the quasielastic intensity, a dynamic change is firstly observed in a highly hydrated protein, around 70 °C, and interpreted in relation with the denaturation mechanism of the protein. The use of heavy water and partly deuterated trehalose gives clear information on protein–trehalose interactions in the native state of lysozyme (at room temperature) and during the thermal denaturation process of lysozyme. At room temperature, it was found that trehalose is preferentially excluded from the protein surface, and has a main effect on the tetrahedral local order of water molecules corresponding to a stiffening of the H-bond network in the solvent. The consequence is a significant reduction of the amplitude of fast relaxational motions, inducing a less marked dynamic transition shifted toward the high temperatures. Upon heating, interaction between trehalose and lysozyme is detected during the solvent penetration within the protein, i.e., while the native globular state softens into a molten globule (MG) state. Addition of trehalose reduces the protein flexibility in the MG state, improving the structural stability of the protein, and inhibiting the protein aggregation.

  19. FORMATION OF PROTOPLASTS FROM STREPTOCOCCUS FAECALIS BY LYSOZYME1

    PubMed Central

    Bibb, William R.; Straughn, W. R.

    1962-01-01

    Bibb, William R. (University of North Carolina, Chapel Hill) and W. R. Straughn. Formation of protoplasts from Streptococcus faecalis by lysozyme. J. Bacteriol. 84:1094–1098. 1962.—Incubation of whole cells of Streptococcus faecalis F24 in the presence of the crystalline egg-white lysozyme and appropriate sucrose concentration resulted in the formation of discrete spherical structures. On dilution, these osmotically fragile structures lysed immediately. Methyl pentose determinations on isolated cell walls and protoplast membranes verified the presence of rhamnose in the cell walls and its essentially complete absence in protoplast membranes. Cell walls were rendered soluble by lysozyme. After lysozyme treatment of cell walls, 96% of the rhamnose present was not sedimented by centrifugation at 12,500 × g for 30 min. No cell-wall structures were recognized by phasecontrast or electron microscopy. After direct lysis of whole cells of S. faecalis F24 by lysozyme, protoplast membranes were isolated. It is concluded that, in the strain of group D streptococcus studied, lysozyme effectively removes the cell wall. Images PMID:13968087

  20. A Lysin motif (LysM)-containing protein functions in antibacterial responses of red swamp crayfish, Procambarus clarkii.

    PubMed

    Shi, Xiu-Zhen; Zhou, Jing; Lan, Jiang-Feng; Jia, Yu-Ping; Zhao, Xiao-Fan; Wang, Jin-Xing

    2013-01-01

    Lysin domain (LysM) is a widely spread domain in nature and could bind different peptidoglycans and chitin-like compounds in bacteria and eukaryotes. In plants, Lysin motif containing proteins are one of the major classes of pattern recognition proteins which can recognize GlcNAc-containing glycans and have important functions in plant immunity. However, their functions in animal immunity are still unclear. In this study, a cDNA encoding a LysM containing protein was identified from red swamp crayfish, Procambarus clarkii. The cDNA of PcLysM contained 1200 base pair nucleotides with an open reading frame of 702bp encoding a protein of 233 amino acid residues. The deduced protein had a calculated molecular mass of 25.950kDa and a pI of 6.84. Tissue distribution analysis in mRNA level showed that it was highly expressed in gills, hemocytes, and intestine, and lowly expressed in hearts, hepatopancreas, and stomach. Time course expression pattern analysis showed that PcLysM was upregulated in hemocytes and gills after challenge with Vibrio anguillarum, and it was upregulated at 12h after challenge with Staphylococcus aureus in gills. The recombinant PcLysM could bind to different bacteria, and yeast. Further study revealed that PcLysM could bind to peptidoglycans from different bacteria, and chitin. After PcLysM was knocked down, the upregulation of antimicrobial peptide (AMP) genes (crustins and antilipopolysaccharide factors) was suppressed in response to bacterial infection in gills. These results suggest that PcLysM recognizes different microorganisms through binding to polysaccharides, such as peptidoglycans and chitin and regulates the expression of some antimicrobial peptide genes though unknown pathways and regulates the expression of some antimicrobial peptide genes though unknown pathways. This study might provide a clue to elucidate the roles of PcLysM in the innate immune reaction of crayfish P. clarkii.

  1. Triclinic lysozyme at 0.65 A resolution.

    PubMed

    Wang, Jiawei; Dauter, Miroslawa; Alkire, Randy; Joachimiak, Andrzej; Dauter, Zbigniew

    2007-12-01

    The crystal structure of triclinic hen egg-white lysozyme (HEWL) has been refined against diffraction data extending to 0.65 A resolution measured at 100 K using synchrotron radiation. Refinement with anisotropic displacement parameters and with the removal of stereochemical restraints for the well ordered parts of the structure converged with a conventional R factor of 8.39% and an R(free) of 9.52%. The use of full-matrix refinement provided an estimate of the variances in the derived parameters. In addition to the 129-residue protein, a total of 170 water molecules, nine nitrate ions, one acetate ion and three ethylene glycol molecules were located in the electron-density map. Eight sections of the main chain and many side chains were modeled with alternate conformations. The occupancies of the water sites were refined and this step is meaningful when assessed by use of the free R factor. A detailed description and comparison of the structure are made with reference to the previously reported triclinic HEWL structures refined at 0.925 A (at the low temperature of 120 K) and at 0.95 A resolution (at room temperature).

  2. Interaction mechanism between berberine and the enzyme lysozyme

    NASA Astrophysics Data System (ADS)

    Cheng, Ling-Li; Wang, Mei; Wu, Ming-Hong; Yao, Si-De; Jiao, Zheng; Wang, Shi-Long

    2012-11-01

    In the present paper, the interaction between model protein lysozyme (Lys) and antitumorigenic berberine (BBR) was investigated by spectroscopic methods, for finding an efficient and safe photosensitizer with highly active transient products using in photodynamic therapy study. The fluorescence data shows that the binding of BBR could change the environment of the tryptophan (Trp) residues of Lys, and form a new complex. Static quenching is the main fluorescence quenching mechanism between Lys and BBR, and there is one binding site in Lys for BBR and the type of binding force between them was determined to be hydrophobic interaction. Furthermore, the possible interaction mechanism between BBR and Lys under the photoexcitation was studied by laser flash photolysis method, the results demonstrated that BBR neutral radicals (BBR(-H)•) react with Trp (K = 3.4 × 109 M-1 s-1) via electron transfer to give the radical cation (Trp/NH•+) and neutral radical of Trp (TrpN•). Additionally BBR selectively oxidize the Trp residues of Lys was also observed by comparing the transient absorption spectra of their reaction products. Through thermodynamic calculation, the reaction mechanisms between 3BBR∗ and Trp or Lys were determined to be electron transfer process.

  3. Triclinic lysozyme at 0.65 angstrom resolution.

    SciTech Connect

    Wang, J.; Dauter, M.; Alkire, R.; Joachimiak, A.; Dauter, Z.; Biosciences Division; SAIC-Frederick Inc.; National Cancer Inst.

    2007-01-01

    The crystal structure of triclinic hen egg-white lysozyme (HEWL) has been refined against diffraction data extending to 0.65 {angstrom} resolution measured at 100 K using synchrotron radiation. Refinement with anisotropic displacement parameters and with the removal of stereochemical restraints for the well ordered parts of the structure converged with a conventional R factor of 8.39% and an R{sub free} of 9.52%. The use of full-matrix refinement provided an estimate of the variances in the derived parameters. In addition to the 129-residue protein, a total of 170 water molecules, nine nitrate ions, one acetate ion and three ethylene glycol molecules were located in the electron-density map. Eight sections of the main chain and many side chains were modeled with alternate conformations. The occupancies of the water sites were refined and this step is meaningful when assessed by use of the free R factor. A detailed description and comparison of the structure are made with reference to the previously reported triclinic HEWL structures refined at 0.925 {angstrom} (at the low temperature of 120 K) and at 0.95 {angstrom} resolution (at room temperature).

  4. Dielectric behavior of lysozyme and ferricytochrome-c in water/ethylene-glycol solutions.

    PubMed

    Bonincontro, A; Cinelli, S; Onori, G; Stravato, A

    2004-02-01

    This work deals with a dielectric study at radio frequencies of the influence at room temperature of two organic molecules, known as cryo-protectants, ethylene-glycol and glycerol, on conformational and dynamic properties of two model proteins, lysozyme (lys) from chicken egg-white and ferricytochrome-c (cyt-c) from horse heart. Cyt-c is a compact globular protein whereas lys is composed of two structural domains, separated by the active site cleft. Measurements were carried out at the fixed temperature of 20 degrees C varying the concentration of the cosolvent up to 90% w/w. From the analysis of the dielectric relaxation of the protein solution, the effective hydrodynamic radius and the electric dipole moment of the protein were calculated as a function of the cosolvent concentration. The data show that glycerol does not modify significantly the conformation of both proteins and cyt-c is also stable in the presence of ethylene-glycol. On the contrary ethylene-glycol strongly affects the dielectric response of lysozyme denoting a specific effect on its conformation and dynamics. The data are coherently interpreted hypothesizing that glycol molecule wedges between and separates the two domains of lys making them rotationally independent.

  5. Calcium-binding and structural stability of echidna and canine milk lysozymes.

    PubMed

    Kikuchi, M; Kawano, K; Nitta, K

    1998-10-01

    For echidna and canine milk lysozymes, which were presumed to be the calcium-binding lysozymes by their amino acid sequences, we have quantitated their calcium-binding strength and examined their guanidine unfolding profiles. The calcium-binding constants of echidna and canine lysozymes were determined to be 8.6 x 10(6) M(-1) and 8.9 x 10(6) M(-1) in 0.1 M KCl at pH 7.1 and 20 C, respectively. The unfolding of decalcified canine lysozyme proceeds in the same manner as that of alpha-lactalbumin, through a stable molten globule intermediate. However, neither calcium-bound nor decalcified echidna lysozyme shows a stable molten globule intermediate. This unfolding profile of echidna lysozyme is identical to that of conventional lysozymes and pigeon egg-white lysozyme, avian calcium-binding lysozyme. This result supports the suggestion of Prager and Jolles (Prager EM, Jolles P. 1996. Animal lysozymes c and g: An overview. In: Jolles P, ed. Lysozymes: Model enzymes in biochemistry and biology. Basel-Boston-Berlin: Birkhauzer Verlag. pp 9-31) that the lineage of avian and echidna calcium-binding lysozymes and that of eutherian calcium-binding lysozymes diverged separately from that of conventional lysozymes.

  6. Investigations on the role of a lysozyme from the malaria vector Anopheles dirus during malaria parasite development.

    PubMed

    Lapcharoen, Parichat; Komalamisra, Narumon; Rongsriyam, Yupha; Wangsuphachart, Voranuch; Dekumyoy, Paron; Prachumsri, Jetsumon; Kajla, Mayur K; Paskewitz, Susan M

    2012-01-01

    A cDNA encoding a lysozyme was obtained by rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR) from females of the malaria vector Anopheles dirus A (Diptera: Culicidae). The 623 bp lysozyme (AdLys c-1) cDNA encodes the 120 amino acid mature protein with a predicted molecular mass of 13.4 kDa and theoretical pI of 8.45. Six cysteine residues and a potential calcium binding motif that are present in AdLys c-1 are highly conserved relative to those of c-type lysozymes found in other insects. RT-PCR analysis of the AdLys c-1 transcript revealed its presence at high levels in the salivary glands both in larval and adult stages and in the larval caecum. dsRNA mediated gene knockdown experiments were conducted to examine the potential role of this lysozyme during Plasmodium berghei infection. Silencing of AdLys c-1 resulted in a significant reduction in the number of oocysts as compared to control dsGFP injected mosquitoes.

  7. Antibacterial Silver

    PubMed Central

    Clement, Julia L.; Jarrett, Penelope S.

    1994-01-01

    The antibacterial activity of silver has long been known and has found a variety of applications because its toxicity to human cells is considerably lower than to bacteria. The most widely documented uses are prophylactic treatment of burns and water disinfection. However, the mechanisms by which silver kills cells are not known. Information on resistance mechanisms is apparently contradictory and even the chemistry of Ag+ in such systems is poorly understood. Silver binds to many cellular components, with membrane components probably being more important than nucleic acids. It is difficult to know whether strong binding reflects toxicity or detoxification: some sensitive bacterial strains have been reported as accumulating more silver than the corresponding resistant strain, in others the reverse apparently occurs. In several cases resistance has been shown to be plasmid mediated. The plasmids are reported as difficult to transfer, and can also be difficult to maintain, as we too have found. Attempts to find biochemical differences between resistant and sensitive strains have met with limited success: differences are subtle, such as increased cell surface hydrophobicity in a resistant Escherichia coli. Some of the problems are due to defining conditions in which resistance can be observed. Silver(I) has been shown to bind to components of cell culture media, and the presence of chloride is necessary to demonstrate resistance. The form of silver used must also be considered. This is usually water soluble AgNO3, which readily precipitates as AgCl. The clinically preferred compound is the highly insoluble silver sulfadiazine, which does not cause hypochloraemia in burns. It has been suggested that resistant bacteria are those unable to bind Ag+ more tightly than does chloride. It may be that certain forms of insoluble silver are taken up by cells, as has been found for nickel. Under our experimental conditions, silver complexed by certain ligands is more cytotoxic

  8. Structural characterization of new Schiff bases of sulfamethoxazole and sulfathiazole, their antibacterial activity and docking computation with DHPS protein structure.

    PubMed

    Mondal, Sudipa; Mandal, Santi M; Mondal, Tapan Kumar; Sinha, Chittaranjan

    2015-01-01

    New Schiff bases (1, 2) of substituted salicylaldehydes and sulfamethoxazole (SMX)/sulfathiazole (STZ) are synthesized and characterized by elemental analysis and spectroscopic data. Single crystal X-ray structure of one of the compounds (E)-4-((3,5-dichloro-2-hydroxybenzylidene)amino)-N-(5-methylisoxazol-3-yl)benzenesulfonamide (1c) has been determined. Antimicrobial activities of the Schiff bases and parent sulfonamides (SMX, STZ) have been examined against several Gram-positive and Gram-negative bacteria and sulfonamide resistant pathogens; the lowest MIC is observed for (E)-4-((3,5-dichloro-2-hydroxybenzylidene)amino)-N-(thiazol-2-yl)benzene sulfonamide (2c) (8.0 μg mL(-1)) and (E)-4-((3,5-dichloro-2-hydroxybenzylidene)amino)-N-(5-methylisoxazol-3-yl)benzene sulfonamide (1c) (16.0 μg mL(-1)) against sulfonamide resistant pathogens. DFT optimized structures of the Schiff bases have been used to carry out molecular docking studies with DHPS (dihydropteroate synthase) protein structure (downloaded from Protein Data Bank) using Discovery Studio 3.5 to find the most preferred binding mode of the ligand inside the protein cavity. The theoretical data have been well correlated with the experimental results. Cell viability assay and ADMET studies predict that 1c and 2c have good drug like characters.

  9. The interaction of flavonoid-lysozyme and the relationship between molecular structure of flavonoids and their binding activity to lysozyme.

    PubMed

    Yang, Ran; Yu, Lanlan; Zeng, Huajin; Liang, Ruiling; Chen, Xiaolan; Qu, Lingbo

    2012-11-01

    In this work, the interactions of twelve structurally different flavonoids with Lysozyme (Lys) were studied by fluorescence quenching method. The interaction mechanism and binding properties were investigated. It was found that the binding capacities of flavonoids to Lys were highly depend on the number and position of hydrogen, the kind and position of glycosyl. To explore the selectivity of the bindings of flavonoids with Lys, the structure descriptors of the flavonoids were calculated under QSAR software package of Cerius2, the quantitative relationship between the structures of flavonoids and their binding activities to Lys (QSAR) was performed through genetic function approximation (GFA) regression analysis. The QSAR regression equation was K(A) = 37850.460 + 1630.01Dipole +3038.330HD-171.795MR. (r = 0.858, r(CV)(2) = 0.444, F((11,3)) = 7.48), where K(A) is binding constants, Dipole, HD and MR was dipole moment, number of hydrogen-bond donor and molecular refractivity, respectively. The obtained results make us understand better how the molecular structures influencing their binding to protein which may open up new avenues for the design of the most suitable flavonoids derivatives with structure variants.

  10. Milk-derived proteins and peptides of potential therapeutic and nutritive value.

    PubMed

    Zimecki, Michal; Kruzel, Marian L

    2007-01-01

    Milk and colostrum are rich in proteins and peptides which play a crucial role in development of the immune system in mammalian offspring. Immunotropic properties of these compounds prompted investigators to search for their utility in prevention and therapy of various disorders in humans. The following constituents of milk are of particular interest: 1) Lactoferrin (LF)--exhibits antibacterial, antifungal, antiviral, antiparasite and antitumor activities. It is protective with regard to intestinal epithelium, promotes bone growth and accelerates recovery of the immune system function in immunocompromised animal; 2) A Proline-Rich Polypeptide (PRP) shows a variety of immunotropic functions, including promotion of T-cell maturation and inhibition'of autoimmune disorders. PRP was recently found to improve or stabilize the Instrumental Activity of Daily Living status in Alzheimer's disease patients. 3) Casein--has been protective in experimental bacteremia by eliciting myelopoiesis. Casein hydrolyzates were also protective in diabetic animals, reduced the tumor growth and diminished colicky symptoms in infants. Casein-derived peptides have been found to have antihypertensive effects. Glycomacropeptide (GMP)--a peptide derived from kappa casein, exhibits antibacterial and antithrombotic activities. 4) Alpha lactalbumin (LA)--demonstrates antiviral, antitumor and anti-stress properties. LA-enriched diets were anxiolytic, lowered blood pressure in rats, prevented diarrhea and led to a better weight gain in malnourished children. 5) Lysozyme--is effective in treatment of periodentitis and prevention of tooth decay. Milk enriched in lysozyme was used in feeding premature infants suffering from concomitant diseases. 6) Lactoperoxidase--shows antibacterial properties. In conclusion, milk-derived proteins and peptides are bio-accessible and safe for the prevention and treatment of numerous disorders in humans.

  11. Impurity effects on orientation of lysozyme crystals nucleated on fatty acid thin films

    NASA Astrophysics Data System (ADS)

    Kubo, T.; Hondoh, H.; Nakada, T.

    2008-04-01

    Commercially available lysozyme samples that have different lot numbers (E02Z04 and E05802) were crystallized on fatty acid thin films. The orientation of lysozyme crystals nucleated on the films was investigated by atomic force microscopy and optical microscopy. The numbers of lysozyme crystals with specific planes parallel to the films are different. In other words, the impurities contained in commercial lysozyme significantly affect the orientation of lysozyme crystals. Detailed analysis of the orientation distribution of the lysozyme crystals nucleated from the purified sample showed that acetic acid is one of the substances promote the epitaxy.

  12. Lytic sensitivity of Actinobacillus actinomycetemcomitans Y4 to lysozyme.

    PubMed Central

    Iacono, V J; Boldt, P R; MacKay, B J; Cho, M I; Pollock, J J

    1983-01-01

    The ability of both human and hen egg white lysozymes to lyse Actinobacillus actinomycetemcomitans Y4 was investigated. Lysis was followed optically at 540 nm by measuring the percent reduction in turbidity of freshly harvested log-phase cells suspended in Tris-maleate buffers within a wide range of pH (5.2 to 8.5) and molarity (0.01 to 0.2 M) and containing various amounts of enzyme and EDTA. In several instances, treated microorganisms were subsequently examined in thin sections by electron microscopy. Reductions in turbidity and clearing of suspensions occurred with small amounts of lysozyme (less than 1 microgram) under relatively alkaline conditions and at low ionic strength and in the presence of small amounts of EDTA (greater than 0.01 mM). Under the most alkaline conditions, EDTA alone effected turbidity reductions similar to those observed in the presence of lysozyme, which suggested that EDTA not only increased outer membrane permeability but also caused cell lysis. Ultrastructural analysis did not always correspond to turbidimetric observations. Cell lysis was virtually complete in suspensions containing both lysozyme and EDTA. However, in contrast to turbidimetric findings, a significant percentage of cells (greater than 25%) was lysed in the presence of lysozyme alone. Furthermore, significant damage occurred in the presence of EDTA alone. Spheroplast-like cell ghosts were present which surrounded condensed cytoplasm or relatively clear spaces. These findings further support the concept of the requirement for electron microscopy to assess lytic damage in addition to turbidimetric and biochemical methods. Our results are the first to demonstrate the remarkable sensitivity of A. actinomycetemcomitans Y4 to lysozyme and to show that EDTA not only affects outer membrane permeability but effects cell lysis, possibly through activation of autolytic enzymes at the cytoplasmic membrane. The exquisite sensitivity of A. actinomycetemcomitans Y4 to lysis could be

  13. Link between the hydration enthalpy of lysozyme and the density of its hydration water: Electrostriction.

    PubMed

    Danielewicz-Ferchmin, Irena; Ferchmin, A Ryszard

    2010-10-07

    Hydration shells around proteins in solution are on average denser than bulk water. Variations in enthalpy are observed during hydration/dehydration of proteins. To explain consistently those phenomena, a common mechanism-electrostriction-underlying the mechanical and contributing to thermal effects is proposed. The mean mass density of the hydration shell of lysozyme derived from the neutron and X-ray scattering is explained as following the compression of water in the fields of the order of 10(9) V m(-1) due to the charged sites at the boundary of the protein. The mean enthalpy of mixing ΔH(mean) of lysozyme in water calculated on the basis of the measured mean mass density falls in the middle of the values of the enthalpy of mixing ΔH(mix) observed in sorption experiments. This testifies that ΔH(mix) is due in part to the work done by the electrostriction pressure in hydration shell regions situated in high electric fields. The dependence of the sorption enthalpy of exemplary proteins on the number of adsorbed H(2)O molecules is also described in terms of electrostriction.

  14. The Effect of Solution Thermal History on Chicken Egg White Lysozyme Nucleation

    NASA Technical Reports Server (NTRS)

    Burke, Michael W.; Judge, Russell A.; Pusey, Marc L.; Rose, M. Franklin (Technical Monitor)

    2000-01-01

    Proteins are highly flexible molecules and often exhibit defined conformational changes in response to changes in the ambient temperature. Chicken egg white lysozyme has been previously shown to undergo an apparent structural change when warmed above the tetragonal/orthorhombic phase transition temperature. This is reflected by a change in the habit of the tetragonal and orthorhombic crystals so formed. In this study we show that possible conformational changes induced by heating are stable and apparently non- reversible by simple cooling. Exposure of protein solutions to temperatures above the phase change transition temperature, before combining with precipitant solution to begin crystallization, reduces final crystal numbers. Protein that is briefly warmed to 37 C, then cooled shows no sign of reversal to the unheated nucleation behavior even after storage for 4 weeks at 4 C. The change in nucleation behavior of tetragonal lysozyme crystals, attributed to a structural shift, occurs faster the greater the exposure to temperature above the equi-solubility point for the two phases. Heating for 2 h at 48 C reduces crystal numbers by 20 fold in comparison to the same solution heated for the same time at 30 C. Thermal treatment of solutions is therefore a possible tool to reduce crystal numbers and increase crystal size. The effects of a protein's previous thermal history are now shown to be a potentially critical factor in subsequent macromolecule crystal nucleation and growth studies.

  15. The Effect of Solution Thermal History on Chicken Egg White Lysozyme Nucleation

    NASA Technical Reports Server (NTRS)

    Burke, Michael W.; Judge, Russell A.; Pusey, Marc L.

    2001-01-01

    Proteins are highly flexible molecules and often exhibit defined conformational changes in response to changes in the ambient temperature. Chicken egg white lysozyme has been previously shown to undergo an apparent structural change when warmed above the tetragonal/orthorhombic phase transition temperature. This is reflected by a change in the habit of the tetragonal and orthorhombic crystals so formed. In this study, we show that possible conformational changes induced by heating are stable and apparently non-reversible by simple cooling. Exposure of protein solutions to temperatures above the phase change transition temperature, before combining with precipitant solution to begin crystallization, reduces final crystal numbers. Protein that is briefly warmed to 37 C, then cooled shows no sign of reversal to the unheated nucleation behavior even after storage for four weeks at 4 C. The change in nucleation behavior of tetragonal lysozyme crystals, attributed to a structural shift, occurs faster the greater the exposure to temperature above the equi-solubility point for the two phases. Heating for 2 hours at 48 C reduces crystal numbers by 20 fold in comparison to the same solution heated for the same time at 30 C. Thermal treatment of solutions is therefore a possible tool to reduce crystal numbers and increase crystal size. The effects of a protein's previous thermal history are now shown to be a potentially critical factor in subsequent macromolecule crystal nucleation and growth studies.

  16. NMR-based localization of ions involved in salting out of hen egg white lysozyme.

    PubMed

    Poznański, Jarosław

    2006-01-01

    NaCl-induced aggregation of hen egg white lysozyme (HEWL) was monitored by NMR spectroscopy. Small, but significant, changes induced by salt addition in TOCSY spectra were attributed to the effect of local reorganization of protein backbone upon ion binding. Salt-induced variations in HN and H alpha chemical shifts were mapped on the HEWL 3D structure which allowed the construction of a scheme of the spatial localization of potential ion binding sites. It was found that in a 0.5 M NaCl solution six chloride anions and at least one sodium cation are bound to preferred sites on the HEWL surface.

  17. Binding patterns and structure-affinity relationships of food azo dyes with lysozyme: a multitechnique approach.

    PubMed

    Peng, Wei; Ding, Fei; Peng, Yu-Kui; Jiang, Yu-Ting; Zhang, Li

    2013-12-18

    Food dyes serve to beguile consumers: they are often used to imitate the presence of healthful, colorful food produce such as fruits and vegetables. But considering the hurtful impact of these chemicals on the human body, it is time to thoroughly uncover the toxicity of these food dyes at the molecular level. In the present contribution, we have examined the molecular reactions of protein lysozyme with model food azo compound Color Index (C.I.) Acid Red 2 and its analogues C.I. Acid Orange 52, Solvent Yellow 2, and the core structure of azobenzene using a combination of biophysical methods at physiological conditions. Fluorescence, circular dichroism (CD), time-resolved fluorescence, UV-vis absorption as well as computer-aided molecular modeling were used to analyze food dye affinity, binding mode, energy transfer, and the effects of food dye complexation on lysozyme stability and conformation. Fluorescence emission spectra indicate complex formation at 10(-5) M dye concentration, and this corroborates time-resolved fluorescence results showing the diminution in the tryptophan (Trp) fluorescence mainly via a static type (KSV = 1.505 × 10(4) M(-1)) and Förster energy transfer. Structural analysis displayed the participation of several amino acid residues in food dye protein adducts, with hydrogen bonds, π-π and cation-π interactions, but the conformation of lysozyme was unchanged in the process, as derived from fluorescence emission, far-UV CD, and synchronous fluorescence spectra. The overall affinity of food dye is 10(4) M(-1) and there exists only one kind of binding domain in protein for food dye. These data are consistent with hydrophobic probe 8-anilino-1-naphthalenesulfonic acid (ANS) displacement, and molecular modeling manifesting the food dye binding patch was near to Trp-62 and Trp-63 residues of lysozyme. On the basis of the computational analyses, we determine that the type of substituent on the azobenzene structure has a powerful influence on the

  18. THz time scale structural rearrangements and binding modes in lysozyme-ligand interactions.

    PubMed

    Woods, K N

    2014-03-01

    Predicting the conformational changes in proteins that are relevant for substrate binding is an ongoing challenge in the aim of elucidating the functional states of proteins. The motions that are induced by protein-ligand interactions are governed by the protein global modes. Our measurements indicate that the detected changes in the global backbone motion of the enzyme upon binding reflect a shift from the large-scale collective dominant mode in the unbound state towards a functional twisting deformation that assists in closing the binding cleft. Correlated motion in lysozyme has been implicated in enzyme function in previous studies, but detailed characterization of the internal fluctuations that enable the protein to explore the ensemble of conformations that ultimately foster large-scale conformational change is yet unknown. For this reason, we use THz spectroscopy to investigate the picosecond time scale binding modes and collective structural rearrangements that take place in hen egg white lysozyme (HEWL) when bound by the inhibitor (NAG)3. These protein thermal motions correspond to fluctuations that have a role in both selecting and sampling from the available protein intrinsic conformations that communicate function. Hence, investigation of these fast, collective modes may provide knowledge about the mechanism leading to the preferred binding process in HEWL-(NAG)3. Specifically, in this work we find that the picosecond time scale hydrogen-bonding rearrangements taking place in the protein hydration shell with binding modify the packing density within the hydrophobic core on a local level. These localized, intramolecular contact variations within the protein core appear to facilitate the large cooperative movements within the interfacial region separating the α- and β- domain that mediate binding. The THz time-scale fluctuations identified in the protein-ligand system may also reveal a molecular mechanism for substrate recognition.

  19. Specific heat of hydrated lysozyme, water's contribution to its dynamics, and criteria for glass formation of biomaterials.

    PubMed

    Tombari, Elpidio; Johari, G P

    2013-09-14

    Previous studies of the dynamics of hydrated proteins had shown a feature resembling an exceptionally broad glass-softening endotherm. Its onset temperature, denoted as T(g), was indefinable in one calorimetric study of hydrated lysozyme and was in the 148-218 K range in another study, depending upon hydration. Other methods reported this T(g) as ~170 K. We argue that glass-formation of biomaterials should be studied by measuring a property on both the cooling and heating paths and it should be ascertained (i) that there is thermal hysteresis of the measured property, (ii) that the real and imaginary components of a dynamic property obey the Kramers-Kronig relations, and (iii) that there is an effect of annealing that is consistent with the glass phenomenology. We report the real and imaginary components of the dynamic specific heat, C(p)' and C(p)", of dry and two hydrated lysozyme samples on the cooling and the heating paths as well as the effects of annealing and changing the frequency. For the most hydrated (34.6 g water per 100 g lysozyme) sample, C(p,app) does not show thermal hysteresis in the 160-230 K range, C(p)' varies in a sigmoid-shape manner with T while C(p)" remains close to zero, and there is no effect of annealing. We interpret these findings in terms of continuous development of ice-like aggregates of immobile H2O as more H-bonds form on cooling, and continuous deterioration of the aggregates on heating. As the equilibrium constant between the aggregates and mobile H2O increases on cooling, configurational degrees of freedom of H2O molecules and lysozyme segments decrease. Consequently, the net change in enthalpy is small but the change in C(p) is large. Mobility of the lysozyme segments still depends upon the mobility of H2O molecules.

  20. Specific heat of hydrated lysozyme, water's contribution to its dynamics, and criteria for glass formation of biomaterials

    NASA Astrophysics Data System (ADS)

    Tombari, Elpidio; Johari, G. P.

    2013-09-01

    Previous studies of the dynamics of hydrated proteins had shown a feature resembling an exceptionally broad glass-softening endotherm. Its onset temperature, denoted as Tg, was indefinable in one calorimetric study of hydrated lysozyme and was in the 148-218 K range in another study, depending upon hydration. Other methods reported this Tg as ˜170 K. We argue that glass-formation of biomaterials should be studied by measuring a property on both the cooling and heating paths and it should be ascertained (i) that there is thermal hysteresis of the measured property, (ii) that the real and imaginary components of a dynamic property obey the Kramers-Kronig relations, and (iii) that there is an effect of annealing that is consistent with the glass phenomenology. We report the real and imaginary components of the dynamic specific heat, Cp' and Cp″, of dry and two hydrated lysozyme samples on the cooling and the heating paths as well as the effects of annealing and changing the frequency. For the most hydrated (34.6 g water per 100 g lysozyme) sample, Cp,app does not show thermal hysteresis in the 160-230 K range, Cp' varies in a sigmoid-shape manner with T while Cp″ remains close to zero, and there is no effect of annealing. We interpret these findings in terms of continuous development of ice-like aggregates of immobile H2O as more H-bonds form on cooling, and continuous deterioration of the aggregates on heating. As the equilibrium constant between the aggregates and mobile H2O increases on cooling, configurational degrees of freedom of H2O molecules and lysozyme segments decrease. Consequently, the net change in enthalpy is small but the change in Cp is large. Mobility of the lysozyme segments still depends upon the mobility of H2O molecules.

  1. A three-way junction aptasensor for lysozyme detection.

    PubMed

    Xia, Yunfeng; Gan, Siwen; Xu, Qinghao; Qiu, Xiaowen; Gao, Peiyi; Huang, Shasheng

    2013-01-15

    A well-designed three-way junction (TWJ) aptasensor for lysozyme detection was developed based on target-binding-induced conformational change of aptamer-complementary DNA (cDNA) as probe. A ferrocene (Fc)-tagged cDNA is partially hybridized with an anti-lysozyme aptamer to form a folded structure where there is a coaxial stacking of two helices and the third one at an acute angle. In addition, the fabrication of the sensor was achieved via the single-step method, which offered a good condition for sensing. In the absence of lysozyme, electron transfer (eT), through the coaxial two helices called "conductive path", is allowed between Fc-labeled moiety and the electrode. The binding of lysozyme to the aptamer blocks eT, leading to diminished redox signal. This aptasensor with an instinct signal attenuation factor shows a high sensitivity to lysozyme, and the response data is fitted by nonlinear least-squares to Hill equation. Detection limit is 0.2nM with a dynamic range extending to 100nM. Compared with existing electrochemical impedance spectroscopy (EIS)-based approaches, TWJ-DNA aptasensor was demonstrated to be more specific for detection and simpler for regeneration procedure.

  2. Influence of pressure on the low-frequency vibrational modes of lysozyme and water: a complementary inelastic neutron scattering and molecular dynamics simulation study.

    PubMed

    Lerbret, Adrien; Hédoux, Alain; Annighöfer, Burkhard; Bellissent-Funel, Marie-Claire

    2013-02-01

    We performed complementary inelastic neutron scattering (INS) experiments and molecular dynamics (MD) simulations to study the influence of pressure on the low-frequency vibrational modes of lysozyme in aqueous solution in the 1 atm-6 kbar range. Increasing pressure induces a high-frequency shift of the low-frequency part (<10 meV = 80 cm(-1)) of the vibrational density of states (VDOS), g(ω), of both lysozyme and water that reveals a stiffening of the interactions ascribed to the reduction of the protein and water volumes. Accordingly, high pressures increase the curvature of the free energy profiles of the protein quasiharmonic vibrational modes. Furthermore, the nonlinear influence of pressure on the g(ω) of lysozyme indicates a change of protein dynamics that reflects the nonlinear pressure dependence of the protein compressibility. An analogous dynamical change is observed for water and stems from the distortion of its tetrahedral structure under pressure. Moreover, our study reveals that the structural, dynamical, and vibrational properties of the hydration water of lysozyme are less sensitive to pressure than those of bulk water, thereby evidencing the strong influence of the protein surface on hydration water.

  3. Combination of electrografting and atom-transfer radical polymerization for making the stainless steel surface antibacterial and protein antiadhesive.

    PubMed

    Ignatova, Milena; Voccia, Samuel; Gilbert, Bernard; Markova, Nadya; Cossement, Damien; Gouttebaron, Rachel; Jérôme, Robert; Jérôme, Christine

    2006-01-03

    A two-step "grafting from" method has been successfully carried out, which is based on the electrografting of polyacrylate chains containing an initiator for the atom transfer radical polymerization (ATRP) of 2-(tert-butylamino)ethyl methacrylate (TBAEMA) or copolymerization of TBAEMA with either monomethyl ether of poly(ethylene oxide) methacrylate (PEOMA) or acrylic acid (AA) or styrene. The chemisorption of this type of polymer brushes onto stainless steel surfaces has potential in orthopaedic surgery. These films have been characterized by ATR-FTIR, Raman spectroscopy, atomic force microscopy (AFM), and measurement of contact angles of water. The polymer formed in solution by ATRP and that one detached on purpose from the surface have been analyzed by size exclusion chromathography (SEC) and (1)H NMR spectroscopy. The strong adherence of the films onto stainless steel has been assessed by peeling tests. AFM analysis has shown that addition of hydrophilic comonomers to the grafted chains decreases the surface roughness. According to dynamic quartz crystal microbalance experiments, proteins (e.g., fibrinogen) are more effectively repelled whenever copolymer brushes contain neutral hydrophilic (PEOMA) co-units rather than negatively charged groups (PAA salt). Moreover, a 2- to 3-fold decrease in the fibrinogen adsorption is observed when TBAEMA is copolymerized with either PEOMA or AA rather than homopolymerized or copolymerized with styrene. Compared to the bare stainless steel surface, brushes of polyTBAEMA, poly(TBAEMA-co-PEOMA) and poly(TBAEMA-co-AA) decrease the bacteria adhesion by 3 to 4 orders of magnitude as revealed by Gram-positive bacteria S. aureus adhesion tests.

  4. Curcumin and kaempferol prevent lysozyme fibril formation by modulating aggregation kinetic parameters.

    PubMed

    Borana, Mohanish S; Mishra, Pushpa; Pissurlenkar, Raghuvir R S; Hosur, Ramakrishna V; Ahmad, Basir

    2014-03-01

    Interaction of small molecule inhibitors with protein aggregates has been studied extensively, but how these inhibitors modulate aggregation kinetic parameters is little understood. In this work, we investigated the ability of two potential aggregation inhibiting drugs, curcumin and kaempferol, to control the kinetic parameters of aggregation reaction. Using thioflavin T fluorescence and static light scattering, the kinetic parameters such as amplitude, elongation rate constant and lag time of guanidine hydrochloride-induced aggregation reactions of hen egg white lysozyme were studied. We observed a contrasting effect of inhibitors on the kinetic parameters when aggregation reactions were measured by these two probes. The interactions of these inhibitors with hen egg white lysozyme were investigated using fluorescence quench titration method and molecular dynamics simulations coupled with binding free energy calculations. We conclude that both the inhibitors prolong nucleation of amyloid aggregation through binding to region of the protein which is known to form the core of the protein fibril, but once the nucleus is formed the rate of elongation is not affected by the inhibitors. This work would provide insight into the mechanism of aggregation inhibition by these potential drug molecules.

  5. The Effect of Solution Conditions on the Nucleation Kinetics of Tetragonal Lysozyme Crystals

    NASA Technical Reports Server (NTRS)

    Judge, Russell A.; Baird, James K.; Pusey, Marc L.

    1998-01-01

    An understanding of protein crystal nucleation rates and the effect of solution conditions upon them, is fundamental to the preparation of protein crystals of the desired size and shape for X-ray diffraction analysis. The ability to predict the effect of supersaturation, temperature, pH and precipitant concentration on the number and size of crystals formed is of great benefit in the pursuit of protein structure analysis. In this study we experimentally examine the effect of supersaturation, temperature, pH and sodium chloride concentration on the nucleation rate of tetragonal chicken egg white lysozyme crystals. In order to do this batch crystallization plates were prepared at given solution concentrations and incubated at three different temperatures over the period of one week. The number of crystals per well with their size and dimensions were recorded and correlated against solution conditions. Duplicate experiments indicate the reproducibility of the technique. Although it is well known that crystal numbers increase with increasing supersaturation, large changes in crystal number were also correlated against solution conditions of temperature, pH and salt concentration over the same supersaturation ranges. Analysis of these results enhance our understanding of the effect of solution conditions such as the dramatic effect that small changes in charge and ionic strength can have on the number of tetragonal lysozyme crystals that form and grow in solution.

  6. Aggregation behavior of novel heptamethine cyanine dyes upon their binding to native and fibrillar lysozyme.

    PubMed

    Vus, Kateryna; Tarabara, Ulyana; Kurutos, Atanas; Ryzhova, Olga; Gorbenko, Galyna; Trusova, Valeriya; Gadjev, Nikolai; Deligeorgiev, Todor

    2017-04-05

    Two newly synthesized symmetrical heptamethine cyanine dyes, AK7-5 and AK7-6, absorbing in the region of low autofluorescence of biological samples, have been tested for their ability to detect proteins aggregated into amyloid fibrils. In aqueous solution these probes possess three absorption bands corresponding to the monomer, dimer and H-aggregate species. The association of the dye with fibrillar lysozyme was followed by the enhancement of the monomer band and the reduction of the H-band. The absorption spectra measured at various fibril concentrations were analyzed in terms of the model allowing for the shift of equilibria between various dye species due to the binding of monomers and dimers of AK7-5 and AK7-6 to amyloid fibrils. The association constants and stoichiometries of the dye-fibril complexation have been evaluated. In contrast to fibrillar lysozyme, the native protein brought about strong J-aggregate formation accompanied by a marked drop in the absorbance of the dye monomer species. Quantum chemical calculations and simple docking studies showed that AK7-5 and AK7-6 monomers can bind to the grooves, running parallel to the fibril axis. Due to their ability to distinguish between the native and fibrillar protein states, the novel cyanines are recommended as complementary to existing amyloid markers.

  7. Effect of the Surface Charge of Artificial Chaperones on the Refolding of Thermally Denatured Lysozymes.

    PubMed

    Huang, Fan; Shen, Liangliang; Wang, Jianzu; Qu, Aoting; Yang, Huiru; Zhang, Zhenkun; An, Yingli; Shi, Linqi

    2016-02-17

    Artificial chaperones are of great interest in fighting protein misfolding and aggregation for the protection of protein bioactivity. A comprehensive understanding of the interaction between artificial chaperones and proteins is critical for the effective utilization of these materials in biomedicine. In this work, we fabricated three kinds of artificial chaperones with different surface charges based on mixed-shell polymeric micelles (MSPMs), and investigated their protective effect for lysozymes under thermal stress. It was found that MSPMs with different surface charges showed distinct chaperone-like behavior, and the neutral MSPM with PEG shell and PMEO2MA hydrophobic domain at high temperature is superior to the negatively and positively charged one, because of the excessive electrostatic interactions between the protein and charged MSPMs. The results may benefit to optimize this kind of artificial chaperone with enhanced properties and expand their application in the future.

  8. Effect of ethanol-water mixture on the structure and dynamics of lysozyme: A fluorescence correlation spectroscopy study

    NASA Astrophysics Data System (ADS)

    Chattoraj, Shyamtanu; Mandal, Amit Kumar; Bhattacharyya, Kankan

    2014-03-01

    Effect of ethanol-water mixture on the hydrodynamic radius (rH) and conformational dynamics of lysozyme has been studied by circular dichroism, emission spectra, and fluorescence correlation spectroscopy. For this purpose, the protein lysozyme is covalently labeled near the active site with a fluorescent probe, alexa 488. The ethanol molecules are sequestered near the hydrophobic tryptophan residues as indicated by the blue shift of the emission maximum of tryptophan. It is observed that both size (rH) and time constant of conformational relaxation (τR) of lysozyme oscillate with increase in ethanol concentration. The rH of the protein fluctuates from 19 Å in the native state, to a minimum of 13 Å, and a maximum of 29 Å. It is proposed that the oscillating behavior arises from competition between mutual interaction among protein, ethanol, and water. The fluorescence intensity fluctuates because of quenching of the fluorescence of the probe (alexa) by the free amino group of certain residues (e.g., tryptophan). Rate of inter-conversion (folding dynamics) between the open (fluorescent) and closed (non-fluorescent) form has been determined and is found to exhibit similar oscillation with variation in ethanol content.

  9. Fluorescence detection of lipid-induced oligomeric intermediates involved in lysozyme "amyloid-like" fiber formation driven by anionic membranes.

    PubMed

    Melo, Ana M; Ricardo, Joana C; Fedorov, Aleksander; Prieto, Manuel; Coutinho, Ana

    2013-03-14

    Recent findings implicate that "amyloid-like" fiber formation by several non-amyloidogenic proteins/peptides can be triggered by negatively charged lipid membranes. In order to elucidate the factors that govern the formation of these structures, the interaction of lysozyme with phosphatidylserine-containing lipid vesicles was studied by steady-state and time-resolved fluorescence measurements. Three consecutive stages in the interaction of Alexa488-fluorescently labeled lysozyme (Lz-A488) with acidic lipid vesicles were identified in ensemble average measurements. The variation of the mean fluorescence lifetime of Lz-A488 as a function of the surface coverage of the liposomes was quantitatively described by a cooperative partition model that assumes that monomeric lysozyme molecules partition into the bilayer surface and reversibly assemble into oligomers with k subunits (k ≥ 6). The global fit to the experimental data covering a wide range of experimental conditions was performed by taking into account electrostatic effects by means of the Gouy-Chapman theory using a single self-consistent pair of parameters (aggregation constant and stoichiometry). The lipid-protein supramolecular assemblies formed at a low lipid/protein molar ratio were further characterized by fluorescence lifetime imaging microscopy at the single-fiber level, which reported that quenched oligomers are the predominant species in these structures.

  10. Immunohistochemical localization of human immunoglobulins and lysozyme in epoxy-embedded lymph nodes: effect of different fixatives and of proteolytic digestion.

    PubMed

    Dell'Orto, P; Viale, G; Colombi, R; Braidotti, P; Coggi, G

    1982-07-01

    The postembedding immunoperoxidase staining technique for the localization of immunoglobulins (light and heavy chains) and of lysozyme has been successfully applied to epoxy-embedded human lymph nodes, after removal of the resin. Glutaraldehyde-containing fixatives appear to be suitable for the immunohistochemical localization of human immunoglobulins and lysozyme, provided that the masked antigenicity of these proteins is recovered by proteolytic digestion of the tissue sections using 0.4% pepsin or 0.1% trypsin. Nonglutaraldehyde-containing fixatives allow the immunolocalization of human immunoglobulins without any enzymatic pretreatment. This study shows that tissues routinely fixed in glutaraldehyde and embedded for ultrastructural investigations are actually suitable for immunohistochemical studies on human immunoglobulins and lysozyme.

  11. In situ study of the state of lysozyme molecules at the very early stage of the crystallization process by small-angle X-ray scattering

    NASA Astrophysics Data System (ADS)

    Marchenkova, M. A.; Volkov, V. V.; Blagov, A. E.; Dyakova, Yu. A.; Ilina, K. B.; Tereschenko, E. Yu.; Timofeev, V. I.; Pisarevsky, Yu. V.; Kovalchuk, M. V.

    2016-01-01

    The molecular state of hen egg white lysozyme in solution has been studied by small-angle X-ray scattering (SAXS) combined with molecular simulation. The addition of a precipitant is shown to change the state of the protein molecules in solution. The SAXS data were processed using the constructed models of different oligomers. Under the crystallization conditions, lysozyme is shown to be present in solution as monomers (96.0%), dimers (1.9%), and octamers (2.1%), whereas tetramers and hexamers are not found. The modeled structure of the octamer is not consistent with the commonly accepted unit cell containing eight lysozyme molecules. Meanwhile, the modeled octamers are well-fitted to the crystal structure and can serve as building blocks in the course of crystal growth.

  12. The solubility of hen egg-white lysozyme

    NASA Technical Reports Server (NTRS)

    Howard, Sandra B.; Twigg, Pamela J.; Baird, James K.; Meehan, Edward J.

    1988-01-01

    The equilibrium solubility of chicken egg-white lysozyme in the presence of crystalline solid state was determined as a function of NaCl concentration, pH, and temperature. The solubility curves obtained represent a region of the lysozyme phase diagram. This diagram makes it possible to determine the supersaturation of a given set of conditions or to achieve identical supersaturations by different combinations of parameters. The temperature dependence of the solubility permits the evaluation of Delta-H of crystallization. The data indicate a negative heat of crystallization for the tetragonal crystal form but a positive heat of crystallization for the high-temperature orthorhombic form.

  13. Preparation and Characterization of Fluorescent Derivatives of Lysozyme

    NASA Technical Reports Server (NTRS)

    Smith, Lori; Pusey, Marc

    1998-01-01

    Fluorescence is one of the most versatile and powerful tools for the study of macromolecules. However, its use in macromolecular crystal growth studies is hampered by the necessity of preparing fluorescent derivatives where the probe does not markedly affect the crystal packing. Alternatively, one can prepare derivatives of limited utility if it is known that they will not affect the specific goals of a given study. We have prepared a number of fluorescent derivatives of chicken egg white lysozyme, covalently attaching fluorescent probes to two different sites on the protein molecule. The first site is the side chain carboxyl group of ASP 101. Amine containing probes such as lucifer yellow, cascade blue, and 5- (2-aminoethyl) aminonapthalene-l-sulfonic acid (EDANS) have been attached using a carbodiimide coupling procedure. ASP 101 lies within the active site cleft, and it is believed that the probes are "buried" within that cleft. This is supported by the fact that all such derivatives have been found to crystallize, with the crystals being fluorescent. Tetragonal crystals of the lucifer yellow derivative have been found to diffract to at least 1.9 A resolution. X-ray diffraction data has been acquired and we are now working on the structure of this derivative. The second group of derivatives is to the N-terminal amine group. The derivatization reaction is performed by using a succinimidyl ester of the probe to be attached. Fluorescent probes such as pyrene acetic acid, 5-carboxyfluorescein, and Oregon green have been attached to this site. We have had little success in crystallizing these derivatives, probably because this site is part of the contact region between the 43 helix chains. However, these sites do not interfere with formation of the 43 helices and the derivatives are suitable for study of their formation in solution. The derivatives are being characterized by steady state and lifetime fluorescence methods, and the presentation will discuss these