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J Blazyk

Publications and source records attributed to J Blazyk.

13 recordsLinked to original sources
PubMed2001

A novel linear amphipathic beta-sheet cationic antimicrobial peptide with enhanced selectivity for bacterial lipids.

All known naturally occurring linear cationic peptides adopt an amphipathic alpha-helical conformation upon binding to lipids as an initial step in the induction of cell leakage. We designed an 18-residue peptide, (KIGAKI)3-NH2, that has no amphipathic character as an alpha-helix but can form a highly amphipathic beta-sheet. When bound to lipids, (KIGAKI)3-NH2 did indeed form a beta-sheet structure as evidenced by Fourier transform infrared and circular dichroism spectroscopy. The antimicrobial activity of this peptide was compared with that of (KIAGKIA)3-NH2, and it was better than that of GMASKAGAIAGKIAKVALKAL-NH2 (PGLa) and (KLAGLAK)3-NH2, all of which form amphipathic alpha-helices when bound to membranes. (KIGAKI)3-NH2 was much less effective at inducing leakage in lipid vesicles composed of mixtures of the acidic lipid, phosphatidylglycerol, and the neutral lipid, phosphatidylcholine, as compared with the other peptides. However, when phosphatidylethanolamine replaced phosphatidylcholine, the lytic potency of PGLa and the alpha-helical model peptides was reduced, whereas that of (KIGAKI)3-NH2 was improved. Fluorescence experiments using analogs containing a single tryptophan residue showed significant differences between (KIGAKI)3-NH2 and the alpha-helical peptides in their interactions with lipid vesicles. Because the data suggest enhanced selectivity between bacterial and mammalian lipids, linear amphipathic beta-sheet peptides such as (KIGAKI)3-NH2 warrant further investigation as potential antimicrobial agents.

Amino Acid Sequence↗
PubMed2001

Effect of magainin, class L, and class A amphipathic peptides on fatty acid spin labels in lipid bilayers.

Magainins and other antimicrobial peptides increase ion flux across the membrane. They may do this by forming some type of pore or by perturbing lipid organization due to peptide lying on the bilayer surface. In order to determine if magainins perturb the lipid sufficiently to permeabilize the bilayer, their effect on the motion of fatty acid and lipid spin labels in phosphatidylcholine/phosphatidylglycerol (PC/PG) lipid vesicles was determined. Their effect was compared to two synthetic peptides, 18L and Ac-18A-NH(2), designed to mimic the naturally occurring classes of lytic (class L) and apolipoprotein (class A) amphipathic helices, respectively. We show that although magainins and 18L both had significant effects on lipid chain order, much greater than Ac-18A-NH(2), there was no correlation between these effects and the relative ability of these three peptide classes to permeabilize PC/PG vesicles in the order magainins=Ac-18A-NH(2) >> 18L. This suggests that the perturbing effects of magainins on lipid chain order at permeabilizing concentrations are not directly responsible for the increased leakage of vesicle contents. The greater ability of the magainins to permeabilize PC/PG vesicles relative to 18L is thus more likely due to formation of some type of pore by magainins. The greater ability of Ac-18A-NH(2) relative to 18L to permeabilize PC/PG vesicles despite its lack of disordering effect must be due to its ability to cause membrane fragmentation. Effects of these peptides on other lipids indicated that the mechanism by which they permeabilize lipid bilayers depends both on the peptide and on the lipid composition of the vesicles.

Amino Acid Sequence↗
PubMed1998

Insertion of magainin into the lipid bilayer detected using lipid photolabels.

We investigated the interaction of the antimicrobial peptides Ala19-magainin 2 amide and magainin 2 amide with lipid using two lipid photolabels, azidobenzoyl galactosylceramide (GalCer-PL) and azidobenzoylamido capryloyl galactosylceramide (GalCer-C8-PL), which position their photosensitive groups near the apolar-polar interface and near the center of the bilayer, respectively. Magainins have been postulated to permeabilize membranes either by inserting in a transmembrane fashion into the bilayer and forming a channel or by binding to the surface of the bilayer and disturbing lipid packing. Evidence for channel formation has been difficult to obtain, possibly because only a fraction of the peptide may form a channel at any one time and because the channels may have a short lifetime. Both photolabels significantly labeled the peptides when bound to acidic phospholipid vesicles. The extent of labeling by GalCer-C8-PL was at least 70% of that by GalCer-PL, indicating that some of the peptide was inserted deeply into the bilayer at least transiently. The extent of labeling of Ala19-magainin 2 amide increased significantly with an increase in the peptide to lipid mole ratio, indicating cooperativity and supporting the channel model. The extent of labeling of this peptide was maximal by 30 s and did not change over 30 min, indicating that peptide insertion is rapid and either that the peptide remains inserted for at least 30 min or that equilibrium between inserted and noninserted peptide is achieved by 30 s. The latter is supported by other studies in the literature. Use of this hydrophobic photolabeling technique has permitted detection of peptide monomers which inserted into the bilayer and/or formed a channel at some time during the labeling procedure.

Animals↗
PubMed1998

A new monofluorinated phosphatidylcholine forms interdigitated bilayers.

16-Fluoropalmitic acid was synthesized from 16-hydroxypalmitic acid using diethylaminosulfur trifluoride. This monofluorinated fatty acid then was used to make 1-palmitoyl-2-[16-fluoropalmitoyl]-phosphatidylcholine (F-DPPC) as a fluorinated analog of dipalmitoylphosphatidylcholine (DPPC). Surprisingly, we found that the phase transition temperature (Tm) of F-DPPC occurs near 50 degrees C, approximately 10 degrees C higher than its nonfluorinated counterpart, DPPC, as judged by both differential scanning calorimetry and infrared spectroscopy. The pretransition observed for DPPC is absent in F-DPPC. A combination of REDOR, rotational-echo double-resonance, and conventional solid-state NMR experiments demonstrates that F-DPPC forms a fully interdigitated bilayer in the gel phase. Electron paramagnetic resonance experiments show that below Tm, the hydrocarbon chains of F-DPPC are more motionally restricted than those of DPPC. X-ray scattering experiments confirm that the thickness and packing of gel phase F-DPPC is similar to that of heptanetriol-induced interdigitated DPPC. F-DPPC is the first phosphoglyceride containing sn-1 and sn-2 ester-linked fatty acyl chains of equal length that spontaneously forms interdigitated bilayers in the gel state in the absence of inducing agents such as alcohols.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1996

Secondary structure and location of a magainin analogue in synthetic phospholipid bilayers.

Magainins are cationic, membrane-active peptides which show broad-spectrum antimicrobial activity. We have investigated the secondary structure and location of an analogue of magainin 2 in synthetic phospholipid bilayers using a combination of Fourier transform infrared (FTIR) spectroscopy and solid-state nuclear magnetic resonance (NMR) spectroscopy. Ala19-magainin 2 amide exhibits both alpha-helix and beta-sheet secondary structures in lipid bilayers containing either dipalmitoylphosphatidylglycerol (DPPG) or a 1:1 molar mixture of DPPG and dipalmitoylphosphatidylcholine (DPPC). The combination of FTIR and solid-state NMR results suggests that there are two populations of peptide. The secondary structure of one population is alpha-helix while that of the other population is beta-sheet. We demonstrate that the solid-state NMR technique, rotational-echo double resonance (REDOR), can be used to measure both intra- and intermolecular dipole-dipole interactions in membrane-bound peptides. Our REDOR experiments indicate that alpha-helical Ala19-magainin 2 amide is bound near the phospholipid head groups.

Amino Acid Sequence↗
PubMed1991

Interactions between the antimicrobial peptide, magainin 2, and Salmonella typhimurium lipopolysaccharides.

Using FT-IR spectroscopy, the effects of magainin 2 on the thermotropic behavior of LPS isolated from wild-type (SL3770) and LPS-mutant strains of Salmonella typhimurium are characterized and compared. The mutant strains include Ra (SL3749), polymyxin-sensitive Rb2(s) (SH5014), polymyxin-resistant Rb2(r) (SH5357) and Rc (HN202) LPS chemotypes, whose polysaccharide chains differ in length but possess an identical number of phosphorylation sites. In all cases, magainin 2 causes a concentration-dependent disordering of the LPS fatty acyl chains. Differences in disordering of LPS correlate more closely with the charge on the LPS molecule (determined by high-resolution 31P NMR) rather than with the length of the LPS sugar side chain, contradicting the currently accepted model for the interaction of cationic antibiotics with the Gram-negative cell envelope.

Amino Acid Sequence↗
PubMed1991

Interactions between magainin 2 and Salmonella typhimurium outer membranes: effect of lipopolysaccharide structure.

The role of the outer membrane and lipopolysaccharide (LPS) in the interaction between the small cationic antimicrobial peptide magainin 2 and the Gram-negative cell envelope was studied by FT-IR spectroscopy. Magainin 2 alters the thermotropic properties of the outer membrane-peptidoglycan complexes from wild-type Salmonella typhimurium and a series of LPS mutants which display differential susceptibility to the bactericidal activity of cationic antibiotics. These results are correlated with the LPS phosphorylation pattern and charge (characterized by high-resolution 31P NMR) and outer membrane lipid composition, and are compared to the bactericidal susceptibility. LPS mutants show a progressive loss of resistance to killing by magainin 2 as the length of the LPS polysaccharide moiety decreases. Disordering of the outer membrane lipid fatty acyl chains by magainin 2, however, depends primarily upon the magnitude of LPS charge rather than the length of the LPS polysaccharide, contradicting the proposal by Weiss et al. [Weiss, J., Beckerdite-Quagiata, S., & Elsbach, P. (1980) J. Clin. Invest. 65, 619-628] that the sugar side chain of LPS shields the negative charges of the outer membrane surface. While disruption of outer membrane structure most likely is not the primary factor leading to cell death, the susceptibility of Gram-negative cells to magainin 2 is associated with factors that facilitate the transport of the peptide across the outer membrane, such as the magnitude and location of LPS charge, the concentration of LPS in the outer membrane, outer membrane molecular architecture, and the presence or absence of the O-antigen side chain.

Anti-Bacterial Agents↗
PubMed1990

Interactions between Salmonella typhimurium lipopolysaccharide and the antimicrobial peptide, magainin 2 amide.

Effects of magainin 2 amide on the phase behavior of Salmonella typhimurium lipopolysaccharide were characterized by FT-IR spectroscopy. This antimicrobial cationic peptide disorders the lipopolysaccharide at molecular ratios of lipopolysaccharide to magainin greater than 4, and can induce a temperature-dependent structural reorientation. The nature of the five phosphate groups of lipopolysaccharide was determined by 31P NMR spectroscopy. At pH 7.4, the net charge on the phosphates is -7. Lipopolysaccharide undoubtedly plays an important role in modulating the interactions of magainin with the gram-negative cell envelope and may act as a molecular sponge to protect the plasma membrane.

Animals↗
PubMed1990

Bactericidal activity of magainin 2: use of lipopolysaccharide mutants.

Salmonella typhimurium and a series of rough lipopolysaccharide mutants derived from it were used as target bacteria to examine the antimicrobial capacity of magainin 2. Magainin 2 demonstrated a dose-related bactericidal activity against the smooth parent strain and the series of lipopolysaccharide mutants. The lipopolysaccharide mutant series showed an ordered increase in sensitivity to the magainin 2 as the depth of the rough lesion in the lipopolysaccharide increased.

Anti-Bacterial Agents↗
PubMed1987

Effect of plasmid RP1 on phase changes in inner and outer membranes and lipopolysaccharide from Acinetobacter calcoaceticus: a Fourier transform infrared study.

The successful transfer of the resistance plasmid RP1 into the Gram-negative bacterium Acinetobacter calcoaceticus resulted in increased resistance of this microorganism to the antibiotics kanamycin and tetracycline. Microorganisms harboring the RP1 plasmid showed altered fatty acid composition in the lipopolysaccharide fraction and increased outer membrane permeability compared to organisms without the plasmid. Thermotropic gel to liquid crystal lipid phase changes were detected in both inner and outer membranes and purified lipopolysaccharide by Fourier transform infrared spectroscopy. The phase transition temperatures observed in the outer membranes and isolated lipopolysaccharide of the plasmid-containing cells were significantly higher than those of the plasmid-free organisms, while little difference was observed for the inner membranes. The plasmid-induced decrease in outer membrane fluidity may play a mediating role in the mechanisms of antibiotic resistance and susceptibility to host immune cells in Gram-negative microorganisms.

Acinetobacter↗
PubMed1985

Correlation between lipid fluidity and tryptic susceptibility of Ca2+-ATPase in sarcoplasmic reticulum membranes.

Lipid fluidity in native and denatured sarcoplasmic reticulum membranes and extracted lipids was monitored between -30 and 30 degrees C using trans-parinaric acid as a fluorescent probe. In addition to a large increase in fluidity between -30 and 0 degree C in each system, a phase change centered near 10 degrees C was observed in the extracted lipids but not in either the native or denatured membranes. A significant change in fluorescence intensity near 15 degrees C was observed in native sarcoplasmic reticulum membranes, however, when trans-parinaric acid was excited by energy transfer from tryptophan residues of the membrane protein. When Ca2+-ATPase was subjected to proteolytic cleavage by trypsin as a function of temperature, a change in susceptibility was detected at about 15-20 degrees C in the native membranes but not in a solubilized preparation. It is proposed that one or more structural changes in the microenvironment of Ca2+-ATPase in the native membrane occur between 15 and 20 degrees C which may be related to the change in apparent activation energy which is observed for this enzyme.

Animals↗