Tilted State Population of Antimicrobial Peptide PGLa Is Coupled to the Transmembrane Potential.

Cationic antimicrobial peptide PGLa gets into close contact with the anionic bacterial cell membrane, facilitating cross-membrane transport phenomena and membrane disruption depending on the concentration. The mechanisms of action are closely associated with the tilted insertion geometry of PGLa. Therefore, we aimed to understand the interaction between the transmembrane potential (TMP) and the orientation of the membrane-bound PGLa helix. Molecular dynamics simulations were performed with TMP, and we found that the PGLa tilt angle relative to the membrane is coupled with the TMP. Elevated TMP increases the population of the tilted state. We observed positive feedback between the tilt angle and the TMP, which occurs due to the electrostatic interaction between the peptidic helix and the Na+ cations at the membrane-water interface. These TMP coupled phenomena can contribute to understanding the direct antimicrobial and adjuvant effects of PGLa in combination with regular antibiotics.

Induced folding of protein-sized foldameric ß-sandwich models with core ß-amino acid residues.

The mimicry of protein-sized ß-sheet structures with unnatural peptidic sequences (foldamers) is a considerable challenge. In this work, the de novo designed betabellin-14 ß-sheet has been used as a template, and αâ†’ß residue mutations were carried out in the hydrophobic core (positions 12 and 19). ß-Residues with diverse structural properties were utilized: Homologous ß(3) -amino acids, (1R,2S)-2-aminocyclopentanecarboxylic acid (ACPC), (1R,2S)-2-aminocyclohexanecarboxylic acid (ACHC), (1R,2S)-2-aminocyclohex-3-enecarboxylic acid (ACEC), and (1S,2S,3R,5S)-2-amino-6,6-dimethylbicyclo[3.1.1]heptane-3-carboxylic acid (ABHC). Six α/ß-peptidic chains were constructed in both monomeric and disulfide-linked dimeric forms. Structural studies based on circular dichroism spectroscopy, the analysis of NMR chemical shifts, and molecular dynamics simulations revealed that dimerization induced ß-sheet formation in the 64-residue foldameric systems. Core replacement with (1R,2S)-ACHC was found to be unique among the ß-amino acid building blocks studied because it was simultaneously able to maintain the interstrand hydrogen-bonding network and to fit sterically into the hydrophobic interior of the ß-sandwich. The novel ß-sandwich model containing 25 % unnatural building blocks afforded protein-like thermal denaturation behavior.

Predicting order and disorder for ß-peptide foldamers in water.

Following a quantitative validation approach, we tested the AMBER ff03 and GAFF force fields with the TIP3P explicit water model in molecular dynamic simulations of ß-peptide foldamers. The test sequences were selected to represent a wide range of folding behavior in water: compact helix, strand mimetic geometry, and the state of disorder. The combination AMBER ff03-TIP3P successfully predicted the experimentally observed conformational properties and reproduced the NOE distances and backbone (3)J coupling data at a good level. GAFF was unable to produce folded structures correctly due to its biased torsion potentials. We can recommend AMBER ff03-TIP3P for simulations involving ß-peptide sequences in aqueous media including ordered and disordered structures.

Rationally designed foldameric adjuvants enhance antibiotic efficacy via promoting membrane hyperpolarization.

The negative membrane potential of bacterial cells influences crucial cellular processes. Inspired by the molecular scaffold of the antimicrobial peptide PGLa, we have developed antimicrobial foldamers with a computer-guided design strategy. The novel PGLa analogues induce sustained membrane hyperpolarization. When co-administered as an adjuvant, the resulting compounds - PGLb1 and PGLb2 - have substantially reduced the level of antibiotic resistance of multi-drug resistant Escherichia coli, Klebsiella pneumoniae and Shigella flexneri clinical isolates. The observed antibiotic potentiation was mediated by hyperpolarization of the bacterial membrane caused by the alteration of cellular ion transport. Specifically, PGLb1 and PGLb2 are selective ionophores that enhance the Goldman-Hodgkin-Katz potential across the bacterial membrane. These findings indicate that manipulating bacterial membrane electrophysiology could be a valuable tool to overcome antimicrobial resistance.

Molecular Dynamics and Metadynamics Insights of 1,4-Dioxane-Induced Structural Changes of Biomembrane Models.

1,4-Dioxane is a cytotoxic B2-type human carcinogen, a serious water pollutant produced solely by industrial activity. The effect of 1,4-dioxane on phospholipid membrane models composed of dipalmitoyl-phosphatidylcholine (DPPC) and its branched isomer (isodipalmitoyl-phosphatidylcholine, IPPC) was investigated using MD simulations. Clear and polluted membranes were compared by membrane parameters such as area per lipid (APL), volume per lipid (VPL), compressibility modulus, membrane thickness, and orderliness of lipid tails. While neat systems significantly differ from each other, the presence of the pollutant has the same effect on both types of lipid membranes. High density of dioxane appears in the vicinity of ester groups, which pushes away lipid headgroups from each other, leading to an overall change in lipid structure: APL and VPL grows, while the orderliness of lipid tails, membrane thickness, and compressibility modulus decrease. Orientational preferences of water and dioxane molecules were also investigated and different membrane regions have been specified according to the stance of water molecules. Free-energy profile for 1,4-dioxane penetration mechanism into DPPC membranes was carried out using metadynamics for two different concentrations of the pollutant (c1 = 7.51 g/dm3, c2 = 75.10 g/dm3), which showed that the higher the concentration is, the lower the free energy of penetration gets. Only a small free-energy barrier was found in the headgroup region and accumulation of dioxane is thermodynamically unfavored in the middle of the bilayer. The penetration mechanism has been described in detail based on the orientational preference of 1,4-dioxane molecules and the free-energy profiles.

Competitive inhibition of TRPV1-calmodulin interaction by vanilloids.

There is enormous interest toward vanilloid agonists of the pain receptor TRPV1 in analgesic therapy, but the mechanisms of their sensory neuron-blocking effects at high or repeated doses are still a matter of debate. Our results have demonstrated that capsaicin and resiniferatoxin form nanomolar complexes with calmodulin, and competitively inhibit TRPV1-calmodulin interaction. These interactions involve the protein recognition interface of calmodulin, which is responsible for all of the cell-regulatory calmodulin-protein interactions. These results draw attention to a previously unknown vanilloid target, which may contribute to the explanation of the paradoxical pain-modulating behavior of these important pharmacons.

Experimental evidence of fluid secretion of rabbit lacrimal gland duct epithelium.

PURPOSE: To investigate the osmotic water permeability of lacrimal gland (LG) duct epithelium by means of calculation of filtration permeability and to investigate LG ductal fluid secretion. METHODS: Experiments were performed on isolated rabbit LG duct segments maintained in short-term culture. Osmotically determined fluid movement or fluid secretion into the closed intraluminal space of cultured LG interlobular ducts was analyzed using video microscopic technique. RESULTS: The end of the LG ducts sealed after overnight incubation forming a closed luminal space. For the calculation of osmotic water permeability, ducts were initially perfused with isotonic HEPES buffered solution, and then with hypotonic HEPES buffered solution. Filtration permeability was calculated from the initial slope of the relative volume increase. Secretory responses to carbachol or to forskolin stimulation were also investigated. Forskolin stimulation resulted in a rapid and sustained secretory response in both solutions. Forskolin-stimulated fluid secretion was completely inhibited by bumetanide both in HEPES buffered and in HCO3 (-)/CO2 buffered solutions, suggesting the central role of Na(+)-K(+)-2Cl(-) cotransporter type 1 (NKCC1). Administration of carbachol initiated a rapid but short secretory response in both HEPES buffered and in HCO3 (-)/CO2 buffered solutions. Atropine completely abolished the carbachol-evoked fluid secretion. CONCLUSIONS: A new method was introduced to investigate LG duct function. Water permeability of rabbit LG duct epithelium was measured by calculating filtration permeability. Fluid secretion of LG duct cells induced by carbachol or forskolin was also demonstrated. These results provide calculated values of lacrimal duct osmotic permeability and direct experimental evidence of LG duct fluid secretion.