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Structure (London, England : 1993) Nov 2003A protocol for the quantitative incorporation of both selenomethionine and selenocysteine into recombinant proteins overexpressed in Escherichia coli is described. This...
A protocol for the quantitative incorporation of both selenomethionine and selenocysteine into recombinant proteins overexpressed in Escherichia coli is described. This methodology is based on the use of a suitable cysteine auxotrophic strain and a minimal medium supplemented with selenium-labeled methionine and cysteine. The proteins chosen for these studies are the cathelin-like motif of protegrin-3 and a nucleoside-diphosphate kinase. Analysis of the purified proteins by electrospray mass spectrometry and X-ray crystallography revealed that both cysteine and methionine residues were isomorphously replaced by selenocysteine and selenomethionine. Moreover, selenocysteines allowed the formation of unstrained and stable diselenide bridges in place of the canonical disulfide bonds. In addition, we showed that NDP kinase contains a selenocysteine adduct on Cys122. This novel selenium double-labeling method is proposed as a general approach to increase the efficiency of the MAD technique used for phase determination in protein crystallography.
Topics: Animals; Antimicrobial Cationic Peptides; Cloning, Molecular; Crystallography, X-Ray; Cysteine; Disulfides; Electrons; Escherichia coli; Mass Spectrometry; Models, Molecular; Nucleoside-Diphosphate Kinase; Oxygen; Plasmids; Protein Conformation; Proteins; Recombinant Proteins; Selenium; Selenocysteine; Selenomethionine; Spectrometry, Mass, Electrospray Ionization; Sulfur; Swine
PubMed: 14604526
DOI: 10.1016/j.str.2003.09.014 -
Antimicrobial Agents and Chemotherapy Mar 2008Rhesus macaque theta-defensins (RTDs) are unique macrocyclic antimicrobial peptides. The three RTDs (RTD 1-3), isolated from macaque leukocytes, have broad-spectrum...
Rhesus macaque theta-defensins (RTDs) are unique macrocyclic antimicrobial peptides. The three RTDs (RTD 1-3), isolated from macaque leukocytes, have broad-spectrum antimicrobial activities in vitro and share certain structural features with acyclic porcine protegrins, which are microbicidal peptides of the cathelicidin family. To understand the structural features that confer the respective cytocidal properties to theta-defensins and protegrins, we determined and compared the biological properties of RTD 1-3 and protegrin 1 (PG-1) in assays for antimicrobial activity, bacterial membrane permeabilization, and toxicity to human cells. RTD 1-3 and PG-1 had similar microbicidal potencies against Escherichia coli, Staphylococcus aureus, and Candida albicans in low-ionic-strength (10 mM) buffers at pH 7.4. The inclusion of physiologic sodium chloride partially inhibited the microbicidal activities of the RTDs, and the degree of inhibition depended on the buffer used in the assay. Similarly, the inclusion of 10% normal human serum partially antagonized the bactericidal activities of all four peptides. In contrast, the microbicidal activities of PG-1 and RTD 1-3 against E. coli were unaffected by physiologic concentrations of calcium chloride and magnesium chloride. Treatment of E. coli ML35 cells with RTD 1-3 or PG-1 rapidly rendered the bacteria permeable to omicron-nitrophenyl-beta-D-galactopyranoside, and this was accompanied by the rapid entry of the RTDs. Finally, although PG-1 was toxic to human fibroblasts and caused a marked lysis of erythrocytes, the RTDs were not cytotoxic or hemolytic. Thus, compared to PG-1, RTD 1-3 possess substantially greater cytocidal selectivity against microbes. Surprisingly, the low cytotoxicity of the RTDs did not depend on the peptides' cyclic conformation.
Topics: Animals; Anti-Infective Agents; Candida albicans; Cell Line; Cell Membrane Permeability; Colony Count, Microbial; Defensins; Escherichia coli; Fibroblasts; Humans; Macaca mulatta; Microbial Sensitivity Tests; Peptides, Cyclic; Staphylococcus aureus
PubMed: 18160518
DOI: 10.1128/AAC.01090-07 -
The FEBS Journal Dec 2014
Topics: Animals; Antimicrobial Cationic Peptides
PubMed: 25387882
DOI: 10.1111/febs.13143 -
European Journal of Biochemistry Feb 2002Gomesin is the first peptide isolated from spider exhibiting antimicrobial activities. This highly cationic peptide is composed of 18 amino-acid residues including four...
Gomesin is the first peptide isolated from spider exhibiting antimicrobial activities. This highly cationic peptide is composed of 18 amino-acid residues including four cysteines forming two disulfide linkages. The solution structure of gomesin has been determined using proton two-dimensional NMR (2D-NMR) and restrained molecular dynamics calculations. The global fold of gomesin consists in a well-resolved two-stranded antiparallel betasheet connected by a noncanonical betaturn. A comparison between the structures of gomesin and protegrin-1 from porcine and androctonin from scorpion outlines several common features in the distribution of hydrophobic and hydrophilic residues. The N- and C-termini, the betaturn and one face of the betasheet are hydrophilic, but the hydrophobicity of the other face depends on the peptide. The similarities suggest that the molecules interact with membranes in an analogous manner. The importance of the intramolecular disulfide bridges in the biological activity of gomesin is being investigated.
Topics: Amino Acid Sequence; Animals; Anti-Infective Agents; Antimicrobial Cationic Peptides; Blood Proteins; Cystine; Models, Molecular; Molecular Sequence Data; Nuclear Magnetic Resonance, Biomolecular; Protein Structure, Secondary; Sequence Homology, Amino Acid; Solutions; Spiders
PubMed: 11856345
DOI: 10.1046/j.0014-2956.2002.02760.x -
Journal of Animal Science Jun 2018The objective of this study was to investigate the effects of Lactobacillus reuteri LR1, a new strain isolated from the feces of weaned pigs, on the growth performance,... (Randomized Controlled Trial)
Randomized Controlled Trial
The objective of this study was to investigate the effects of Lactobacillus reuteri LR1, a new strain isolated from the feces of weaned pigs, on the growth performance, intestinal morphology, immune responses, and intestinal barrier function in weaned pigs. A total of 144 weaned pigs (Duroc × Landrace × Yorkshire, 21 d of age) with an initial BW of 6.49 ± 0.02 kg were randomly assigned to 3 dietary treatments with 8 replicate pens, each of per treatment and 6 pigs. Pigs were fed a basal diet (CON, controls), the basal diet supplemented with 100 mg/kg olaquindox and 75 mg/kg aureomycin (OA) or the basal diet supplemented with 5 × 1010 cfu/kg L. reuteri LR1 for a 14-d period. At the end of study, the ADG, ADFI, and G:F were calculated, and 1 randomly selected pig from each pen was euthanized for sample collection. The LR1 increased ADG (22.73%, P < 0.05) compared with CON. The villus height of the ileum was increased (P < 0.05) and crypt depth in duodenum was reduced (P < 0.05), along with increased (P < 0.05) villus height to crypt depth ratio of the jejunum and ileum by LR1 compared with CON and OA. LR1 increased (P < 0.05) ileal mucosal content of IL-22 and transforming growth factor-β compared with OA. Compared with CON, LR1 increased (P < 0.05) and OA decreased (P < 0.05) the ileal content of secretory immunoglobulin A (sIgA), and the abundance of transcripts of porcine β-defensin 2 and protegrin 1-5. Compared with CON, LR1 increased (P < 0.05) tight junction protein zonula occludens-1 and occludin transcripts in the mucosa of the jejunum and ileum, and those of mucin-2 in ileal mucosa. The relative expression of toll-like receptor 2 (TLR2) and TLR4 were increased (P < 0.05) in ileal mucosa in pigs fed LR1 compared with CON. In conclusion, these data indicated that dietary LR1 supplementation at 5 × 1010 cfu/kg improved growth performance, intestinal morphology, and intestinal barrier function in weaned pigs.
Topics: Animal Feed; Animals; Chlortetracycline; Diet; Dietary Supplements; Feces; Intestinal Mucosa; Limosilactobacillus reuteri; Probiotics; Swine
PubMed: 29659876
DOI: 10.1093/jas/sky129 -
Biophysical Journal Aug 2009Antimicrobial peptides interact specifically with the membrane of a pathogen and kill the pathogen by releasing its cellular contents. Protegrin-1 (PG-1), a beta-hairpin... (Comparative Study)
Comparative Study
Antimicrobial peptides interact specifically with the membrane of a pathogen and kill the pathogen by releasing its cellular contents. Protegrin-1 (PG-1), a beta-hairpin antimicrobial peptide, is known to exist as a transmembrane monomer in a 1,2-dilauroylphosphatidylcholine (DLPC) bilayer and shows concentration-dependent oligomerization in a 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) bilayer. To examine its structure, dynamics, orientation, and interaction in membranes, we performed comparative molecular dynamics simulations of PG-1 monomer and dimer in DLPC and POPC bilayers for a total of 840 ns. The PG-1 monomer exhibits larger tilting in DLPC than in POPC due to a hydrophobic mismatch. PG-1 tilting is dependent on its rotation angle. The specific orientation of PG-1 in membranes is governed by the interactions of its aromatic residues with lipid headgroups. The calculated (15)N and (13)CO chemical shifts of Val(16) in DLPC reveal that there are different sets of tilt and rotation angles that satisfy the experimental values reasonably, suggesting that more experiments are needed to determine its orientation. The dimer simulations show that the dimer interface is better preserved in POPC than in DLPC because POPC's greater hydrophobic thickness causes reduced flexibility of the C-terminal strands. Both monomer and dimer simulations show membrane thinning around PG-1, largely due to arginine-lipid interactions.
Topics: Antimicrobial Cationic Peptides; Computer Simulation; Guanidine; Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; Lipid Bilayers; Models, Chemical; Models, Molecular; Nuclear Magnetic Resonance, Biomolecular; Phosphatidylcholines; Protein Multimerization; Protein Stability; Protein Structure, Secondary; Rotation; Water
PubMed: 19651037
DOI: 10.1016/j.bpj.2009.05.029 -
Gut Jun 2003Postoperative morbidity in patients with obstructive jaundice remains high because of increased susceptibility to endotoxin and the inflammatory cascade.
BACKGROUND
Postoperative morbidity in patients with obstructive jaundice remains high because of increased susceptibility to endotoxin and the inflammatory cascade.
AIMS
An experimental study was designed to investigate the efficacy of protegrin peptide IB-367, an antimicrobial positively charged peptide, in neutralising Escherichia coli 0111:B4 lipopolysaccharide (LPS) in bile duct ligated rats.
METHODS
Adult male Wistar rats were injected intraperitoneally with 2 mg/kg E coli 0111:B4 LPS one week after sham operation or bile duct ligation (BDL). Six groups were studied: sham with placebo, sham with 120 mg/kg tazobactam-piperacillin (TZP), sham with 1 mg/kg IB-367, BDL with placebo, BDL with 120 mg/kg TZP, and BDL with 1 mg/kg IB-367.
RESULTS
Main outcome measures were: endotoxin and tumour necrosis factor alpha (TNF-alpha) concentrations in plasma, evidence of bacterial translocation in blood and peritoneum, and lethality. After LPS, TNF-alpha plasma levels were significantly higher in BDL rats compared with sham operated animals. IB-367 caused a significant reduction in plasma endotoxin and TNF-alpha concentrations compared with placebo and TZP treated groups. In contrast, both TZP and IB-367 significantly reduced bacterial growth compared with saline treatment. Finally, LPS induced 60% and 55% lethality in BDL placebo and TZP treated rats and no lethality in sham operated rats, while only IB-367 significantly reduced lethality to 10%.
CONCLUSIONS
By virtue of its dual antimicrobial and antiendotoxin properties, IB-367 could be an interesting compound to inhibit bacterial translocation and endotoxin release in obstructive jaundice.
Topics: Animals; Anti-Infective Agents; Antimicrobial Cationic Peptides; Bacterial Translocation; Bile Ducts; Cholestasis; Disease Models, Animal; Endotoxemia; Escherichia coli Infections; Ligation; Lipopolysaccharides; Male; Peptides; Postoperative Complications; Proteins; Rats; Rats, Wistar; Tumor Necrosis Factor-alpha
PubMed: 12740345
DOI: 10.1136/gut.52.6.874 -
Chemistry (Weinheim An Der Bergstrasse,... Sep 2021The search for novel antimicrobial agents to combat microbial pathogens is intensifying in response to the rapid development of drug resistance to current antibiotic...
The search for novel antimicrobial agents to combat microbial pathogens is intensifying in response to the rapid development of drug resistance to current antibiotic therapeutics. Respiratory failure and septicemia are the leading causes of mortality among hospitalized patients. Here, the development of a novel engineered cyclotide with effective broad-spectrum antibacterial activity against several ESKAPE bacterial strains and clinical isolates is reported. The most active antibacterial cyclotide was extremely stable in serum, showed little hemolytic activity, and provided protection in vivo in a murine model of P. aeruginosa peritonitis. These results highlight the potential of the cyclotide scaffold for the development of novel antimicrobial therapeutic leads for the treatment of bacteremia.
Topics: Animals; Anti-Bacterial Agents; Anti-Infective Agents; Cyclotides; Humans; Mice; Microbial Sensitivity Tests; Pseudomonas aeruginosa
PubMed: 34159664
DOI: 10.1002/chem.202101438 -
Biochimica Et Biophysica Acta Mar 2007All atom molecular dynamics simulations of the 18-residue beta-hairpin antimicrobial peptide protegrin-1 (PG-1, RGGRLCYCRRRFCVCVGR-NH(2)) in a fully hydrated...
All atom molecular dynamics simulations of the 18-residue beta-hairpin antimicrobial peptide protegrin-1 (PG-1, RGGRLCYCRRRFCVCVGR-NH(2)) in a fully hydrated dilauroylphosphatidylcholine (DLPC) lipid bilayer have been implemented. The goal of the reported work is to investigate the structure of the peptide in a membrane environment (previously solved only in solution [R.L. Fahrner, T. Dieckmann, S.S.L. Harwig, R.I. Lehrer, D. Eisenberg, J. Feigon, Solution structure of protegrin-1, a broad-spectrum antimicrobial peptide from porcine leukocytes. Chemistry and Biology, 3 (1996) 543-550]), and to delineate specific peptide-membrane interactions which are responsible for the peptide's membrane binding properties. A novel, previously unknown, "kick" shaped conformation of the peptide was detected, where a bend at the C-terminal beta-strand of the peptide caused the peptide backbone at residues 16-18 to extend perpendicular to the beta-hairpin plane. This bend was driven by a highly persistent hydrogen-bond between the polar peptide side-chain of TYR7 and the unshielded backbone carbonyl oxygen atom of GLY17. The H-bond formation relieves the unfavorable free energy of insertion of polar groups into the hydrophobic membrane core. PG-1 was anchored to the membrane by strong electrostatic binding of the protonated N-terminus of the peptide to the lipid head group phosphate anions. The orientation of the peptide in the membrane, and its influence on bilayer structural and dynamic properties are in excellent agreement with solid state NMR measurements [S. Yamaguchi, T. Hong, A. Waring, R.I. Lehrer, M. Hong, Solid-State NMR Investigations of Peptide-Lipid Interaction and Orientation of a b-Sheet Antimicrobial Peptide, Protegrin, Biochemistry, 41 (2002) 9852-9862]. Importantly, two simulations which started from different initial orientations of the peptide converged to the same final equilibrium orientation of the peptide relative to the bilayer. The kick-shaped conformation was observed only in one of the two simulations.
Topics: Anti-Infective Agents; Antimicrobial Cationic Peptides; Computer Simulation; Hydrogen Bonding; Lipid Bilayers; Models, Molecular; Phosphatidylcholines; Proteins
PubMed: 17254546
DOI: 10.1016/j.bbamem.2006.11.015 -
Biophysical Journal Oct 2006Protegrins (PG) are important in defending host tissues, preventing infection via an attack on the membrane surface of invading microorganisms. Protegrins have powerful...
Protegrins (PG) are important in defending host tissues, preventing infection via an attack on the membrane surface of invading microorganisms. Protegrins have powerful antibiotic abilities, but the molecular-level mechanisms underlying the interactions of their beta-sheet motifs with the membrane are not known. Protegrin-1 (PG-1) is composed of 18 amino acids with a high content of basic residues and two disulfide bonds. Here we focused on the stability of PG-1 at the amphipathic interface in lipid bilayers and on the details of the peptide-membrane interactions. We simulated all-atom models of the PG-1 monomer with explicit water and lipid bilayers composed of both homogeneous POPC (palmitoyl-oleyl-phosphatidylcholine) lipids and a mixture of POPC/POPG (palmitoyl-oleyl-phosphatidylglycerol) (4:1) lipids. We observed that local thinning of the lipid bilayers mediated by the peptide is enhanced in the lipid bilayer containing POPG, consistent with experimental results of selective membrane targeting. The beta-hairpin motif of PG-1 is conserved in both lipid settings, whereas it is highly bent in aqueous solution. The conformational dynamics of PG-1, especially the highly charged beta-hairpin turn region, are found to be mostly responsible for disturbing the membrane. Even though the eventual membrane disruption requires PG-1 oligomers, our simulations clearly show the first step of the monomeric effects. The thinning effects in the bilayer should relate to pore/channel formation in the lipid bilayer and thus be responsible for further defects in the membrane caused by oligomer.
Topics: Antimicrobial Cationic Peptides; Computer Simulation; Lipid Bilayers; Models, Molecular; Peptides; Phosphatidylcholines; Phosphatidylglycerols; Protein Structure, Secondary; Proteins; Water
PubMed: 16861271
DOI: 10.1529/biophysj.106.084046