All posts in Raw-Journal Peptide Science

This study was concerned with the interaction between the cationic antimicrobial peptide, protamine (Ptm) and the cytoplasmic membranes of the gram-negative bacteria Escherichia coli, Salmonella typhimurium and Pseudomonas aeruginosa. The objective of the study was to explain the observed paradox of internalization without permanent disruption of the cell envelope. We carried out Monte Carlo computer simulation of Ptm in an aqueous environment in the presence of ~100 mM NaCl and model membranes consisting of either (65:35) or (75:25) PE:PG molar ratios. The (75:25) model, representative of the gram-negative cytoplasmic membrane, showed that the Ptm center of mass remained at least 7 nm from the membrane surface leading to the prediction that Ptm would not internalize via disruption of the inner membrane.

By using immunoelectron microscopy of Ptm-treated cells, we showed that Ptm internalization to the cytoplasm took place rapidly in the presence or absence of the outer envelope. Ultrastructural examination revealed no obvious morphological changes to cells that were treated with subinhibitory or bactericidal levels of Ptm. Reconstituted phospholipid bilayers were constructed and were unperturbed by Ptm treatment over a wide range of concentrations and applied transmembrane voltages. We conclude that in the cases of the cell envelopes of E. coli, S. typhimurium and P. aeruginosa, Ptm internalized by means independent of the phospholipid bilayer, most likely mediated by one or more membrane proteins such as cation-selective barrel-like proteins. Work is currently underway to test this hypothesis. © 2014 The Authors. Journal of Peptide Science published by John Wiley & Sons, Ltd.

We have shown that for the cationic antimicrobial peptide protamine, there are at least two different mechanisms of action: one involving complete membrane disruption and in the cases of Escherichia coli, Salmonella typhimurium and Pseudomonas aeruginosa, protamine is internalized by means of a mechanism which is independent of the phospholipid bilayer, but most likely mediated by one or more membrane proteins such as cation-selective barrel-like proteins. Work is currently underway to test this hypothesis.

Slc11a1 is an integral membrane protein with 12 putative transmembrane domains and functions as a pH-coupled divalent metal cation transporter. In the present study, the structures of the peptides corresponding to the second and fifth transmembrane domains of Slc11a1 (from 88 to 109 for TMD2 and from 190 to 215 for TMD5) were determined in membrane-mimic environments by CD and NMR techniques. It was demonstrated that TMD2 and TMD5 form an α-helical structure in 30% 2,2,2-trifluoroethanol (TFE) and 40% hexafluoro-2-propanol (HFIP) aqueous solution, respectively. The α-helix of TMD5 displays a less space-occupied face consisting of the residues Ala194, Gly197, Thr201, Ala204 and Gly208. The α-helix is partially unfolded in the N-terminal region when Gly197 is substituted by Val. The unfolding of the helix in the N-terminal part and/or increase in volume at the less space-occupied face of the helix may exert an effect on the arrangement of TMD5 in membrane. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

The second transmembrane domain TMD2 of Slc11a1 was revealed to adopt an α-helical structure in membrane-mimic environment. The fifth transmembrane domain TMD5 of Slc11a1 was found to form an α-helix with a less space-occupied face, and the α-helix is partially unfolded in the N-terminal region when Gly197 is substituted by Val.

Public health of human beings is threatened by superbugs. Novel human beta-defensins, which contribute to host defense against pathogen invasion and innate immune protection, might be a potent natural candidate pool for new antibiotic lead screening. In the present work, we successfully expressed and purified a novel human beta-defensin, DEFB120, using the IMPACT-TWIN system in Escherichia coli and identified the purified homogeneous proteins using MALDI-TOF mass spectrometry. Then, we performed the fundamental studies on the structure and biological functions for the DEFB120 peptide. The recombinant DEFB120 peptide showed wide antimicrobial effects against E. coli, Staphylococcus aureus and Candida albicans strains without significant hemolytic activity. Furthermore, the high lipopolysaccharide (LPS)-binding affinity in vitro indicated that DEFB120 might be associated with the inhibition of LPS-induced inflammatory response. These results may pave a way for exploiting the essential physiological functions of DEFB120 and also for the development of natural antibiotic pools. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

Novel human beta-defensins contribute to host defense against pathogen invasion and innate immune protection. In the present work, we successfully expressed and purified a novel human beta-defensin, DEFB120, using the IMPACT-TWIN system in Escherichia coli. The recombinant DEFB120 peptide showed wide antimicrobial effects against E. coli, Staphylococcus aureus and Candida albicans strains without significant hemolytic activity. Furthermore, the high lipopolysaccharide (LPS)-binding affinity in vitro indicated that DEFB120 might be associated with the inhibition of LPS-induced inflammatory response.

Public health of human beings is threatened by superbugs. Novel human beta-defensins, which contribute to host defense against pathogen invasion and innate immune protection, might be a potent natural candidate pool for new antibiotic lead screening. In the present work, we successfully expressed and purified a novel human beta-defensin, DEFB120, using the IMPACT-TWIN system in Escherichia coli and identified the purified homogeneous proteins using MALDI-TOF mass spectrometry. Then, we performed the fundamental studies on the structure and biological functions for the DEFB120 peptide. The recombinant DEFB120 peptide showed wide antimicrobial effects against E. coli, Staphylococcus aureus and Candida albicans strains without significant hemolytic activity. Furthermore, the high lipopolysaccharide (LPS)-binding affinity in vitro indicated that DEFB120 might be associated with the inhibition of LPS-induced inflammatory response. These results may pave a way for exploiting the essential physiological functions of DEFB120 and also for the development of natural antibiotic pools. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

Novel human beta-defensins contribute to host defense against pathogen invasion and innate immune protection. In the present work, we successfully expressed and purified a novel human beta-defensin, DEFB120, using the IMPACT-TWIN system in Escherichia coli. The recombinant DEFB120 peptide showed wide antimicrobial effects against E. coli, Staphylococcus aureus and Candida albicans strains without significant hemolytic activity. Furthermore, the high lipopolysaccharide (LPS)-binding affinity in vitro indicated that DEFB120 might be associated with the inhibition of LPS-induced inflammatory response.

The influence of aqueous environment on the main-chain conformation (ω₀, ϕ, and ψ dihedral angles) of two model peptoids: N-acetyl-N-methylglycine N’-methylamide (Ac-N(Me)-Gly-NHMe) (1) and N-acetyl-N-methylglycine N’,N’-dimethylamide (Ac-N(Me)-Gly-NMe₂) (2) was investigated by MP2/6-311++G(d,p) method. The Ramachandran maps of both studied molecules with cis and trans configuration of the N-terminal amide bond in the gas phase and in water environment were obtained and all energy minima localized. The polarizable continuum model was applied to estimate the solvation effect on conformation. Energy minima of the Ac-N(Me)-Gly-NHMe and Ac-N(Me)-Gly-NMe₂ have been analyzed in terms of the possible hydrogen bonds and C = O dipole attraction. To validate the theoretical results obtained, conformations of the similar structures gathered in the Cambridge Crystallographic Data Centre were analyzed. Obtained results indicate that aqueous environment in model peptoids 1 and 2 favors the conformation F (ϕ and ψ = −70º, 180º), and additionally significantly increases the percentage of structures with cis configuration of N-terminal amide bond in studied compounds. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

The influence of water on peptoid conformational properties was studied by PCM/MP2 method. The theoretical calculations show that aqueous environment increases the tendency of methylated peptide bond to adopt the cis configuration and distinctly stabilizes conformers with torsion angles corresponding to polyproline helices PPII and PPI.

Use of the 4-pyridylmethyl ester group for side-chain protection of glutamic acid residues in solid-phase peptide synthesis enables switching of the charge state of a peptide from negative to positive, thus making detection by positive ion mode ESI-MS possible. The pyridylmethyl ester moiety is readily removed from peptides in high yield by hydrogenation. Combining the 4-pyridylmethyl ester protecting group with benzyl ester protection reduces the number of the former needed to produce a net positive charge and allows for purification by RP HPLC. This protecting group is useful in the synthesis of highly acidic peptide sequences, which are often beset by problems with purification by standard RP HPLC and characterization by ESI-MS. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

Side-chain protection of glutamic and aspartic acid residues by the 4-pyridylmethyl ester group enables the charge state of a peptide to be switched from negative to positive, thus making detection by positive ion mode ESI-MS possible. This protecting group is useful in the synthesis of highly acidic peptide sequences, which are often beset by problems with purification by standard RP HPLC and characterization by ESI-MS.

Use of the 4-pyridylmethyl ester group for side-chain protection of glutamic acid residues in solid-phase peptide synthesis enables switching of the charge state of a peptide from negative to positive, thus making detection by positive ion mode ESI-MS possible. The pyridylmethyl ester moiety is readily removed from peptides in high yield by hydrogenation. Combining the 4-pyridylmethyl ester protecting group with benzyl ester protection reduces the number of the former needed to produce a net positive charge and allows for purification by RP HPLC. This protecting group is useful in the synthesis of highly acidic peptide sequences, which are often beset by problems with purification by standard RP HPLC and characterization by ESI-MS. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

Side-chain protection of glutamic and aspartic acid residues by the 4-pyridylmethyl ester group enables the charge state of a peptide to be switched from negative to positive, thus making detection by positive ion mode ESI-MS possible. This protecting group is useful in the synthesis of highly acidic peptide sequences, which are often beset by problems with purification by standard RP HPLC and characterization by ESI-MS.