Effect of dimerization on the mechanism of action of aurein 1.2.

The mechanism of action of antimicrobial peptides depends on physicochemical properties such as structure, concentration, and oligomerization. Here, we focused on the effect of dimerization on the mechanism of action of aurein 1.2 (AU). We designed a lysine-linked AU dimer, (AU)2K, and its interaction with membrane mimetics was studied using four biophysical techniques and molecular dynamics simulations. Circular dichroism and molecular dynamics studies showed that AU displayed a typical spectrum for disordered structures in aqueous solution whereas (AU)2K exhibited the typical spectrum of α-helices in a coiled-coil conformation, wherein helices are wrapped around each other. With the addition of large unilamellar vesicles (LUVs), AU adopted an α-helix structure whereas the coiled-coil structure of (AU)2K assumed an extended conformation. Carboxyfluorescein release experiments with LUVs showed that both peptides were able to permeabilize vesicles although the leakage response to increases in peptide concentration differed. Optical microscopy experiments showed that both peptides induced pore opening and the dimer eventually caused the vesicles to burst. Finally, calorimetric traces determined by isothermal titration calorimetry on the LUVs also showed significant differences in peptide-membrane interactions. Together, the results of our study demonstrated that dimerization changes the mechanism of action of AU.

Composition dependence of vesicle morphology and mixing properties in a bacterial model membrane system.

We have determined the mixing properties and lamellar organization of bacterial membrane mimetics composed of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) and -phosphatidylglycerol (POPG) at various molar ratios applying differential scanning calorimetry, small and wide-angle X-ray scattering, as well as optical phase contrast microscopy. Combining the experimental thermodynamic data with a simulation of the liquidus and solidus lines, we were able to construct a phase diagram. Using this approach, we find that the lipids mix in all phases non-ideally in the thermodynamic sense. As expected, pure POPE assembles into multilamellar and pure POPG into unilamellar vesicles, respectively, which are stable within the studied temperature range. In contrast, mixtures of the two components form oligolamellar vesicles consisting of about three to five bilayers. The layers within these oligolamellar liposomes are positionally correlated within the gel phase, but become uncorrelated within the fluid phase exhibiting freely fluctuating bilayers, while the vesicles as a whole remain intact and do not break up into unilamellar forms. X-ray, as well as DSC data, respectively, reveal a miscibility gap due to a lateral phase segregation at POPG concentrations above about 70 mol%, similar to previously reported data on mixtures composed of disaturated PEs and PGs. Hence, the existence of a region of immiscibility is a general feature of PE/PG mixtures and the mixing properties are dominated by PE/PG headgroup interactions, but are largely independent of the composition of the hydrocarbon chains. This is in accordance with a recent theoretical prediction.

Mesoscopic structure in the chain-melting regime of anionic phospholipid vesicles: DMPG.

In a range of low ionic strength, aqueous dispersions of the anionic phospholipid DMPG (dimyristoylphosphatidylglycerol) have a transparent intermediate phase (IP, between T(m)(on) congruent with 20 degrees C and T(m)(off) congruent with 30 degrees C) between the turbid gel and fluid membrane phases, evidenced in turbidity data. Small angle x-ray scattering results on DMPG dispersions show that, besides the bilayer peak present in all phases, a peak corresponding to a mesoscopic structure at approximately 400 A is detected only in IP. The dependence of this peak position on DMPG concentration suggests a correlation in the bilayer plane, consistent with the stability of vesicles in IP. Moreover, observation of giant DMPG vesicles with phase contrast light microscopy show that vesicles "disappear" upon cooling below T(m)(off) and "reappear" after reheating. This further proves that although vesicles cannot be visualized in IP, their overall structure is maintained. We propose that the IP in the melting regime corresponds to unilamellar vesicles with perforations, a model which is consistent with all described experimental observations. Furthermore, the opening of pores across the membrane tuned by ionic strength, temperature, and lipid composition is likely to have biological relevance and could be used in applications for controlled release from nanocompartments.

Thermal transitions of DMPG bilayers in aqueous solution: SAXS structural studies.

Dimyristoylphosphatidylglycerol (DMPG) has been extensively studied as a model for biological membranes, since phosphatidylglycerol is the most abundant anionic phospholipid in prokaryotic cells. At low ionic strengths, this lipid presents a peculiar thermal behavior, with two sharp changes in the light scattering profile, at temperatures named here T(on)(m) and T(off)(m). Structural changes involved in the DMPG thermal transitions are here investigated by small angle X-ray scattering (SAXS), and compared to the results yielded by differential scanning calorimetry (DSC) and electron spin resonance (ESR). The SAXS results show a broad peak, indicating that DMPG is organized in single bilayers, for the range of temperature studied (10-45 degrees C). SAXS intensity shows an unusual effect, starting to decrease at T(on)(m), and presenting a sharp increase at T(off)(m). The bilayer electron density profiles, obtained from modeling the SAXS curves, show a gradual decrease in electron density contrast (attributed to separation between charged head groups) and in bilayer thickness between T(on)(m) and T(off)(m). Results yielded by SAXS, DSC and ESR indicate that a chain melting process starts at T(on)(m), but a complete fluid phase exists only for temperatures above T(off)(m), with structural changes occurring at the bilayer level in the intermediate region.

Postoperative bladder distention: measurement with bladder ultrasonography.

Bladder distention is a common postoperative occurrence. A process improvement project was conducted at a Midwestern Veteran Affairs Medical Center to determine whether a new method for detecting bladder distention, bladder ultrasonography, was more effective than manual palpation in the perianesthesia setting. Data were collected on 494 men over a 9-month period using bladder ultrasonography. Of those patients, 19.4% had postoperative bladder distention with greater than 400 mL of urine. This compared with 1.4% of patients who had bladder distention detected during the previous year using manual palpation. Data from the project supported the use of bladder ultrasonography as being more effective than manual palpation in the assessment of postoperative bladder distention in the PACU.

Probing DMPG vesicle surface with a cationic aqueous soluble spin label.

A small, highly aqueous soluble, deuterated, cationic spin label, 4-trimethylammonium-2,2,6,6-tetramethylpiperidine-d17-1-oxyl iodide (dCAT1), was used to directly monitor the negatively charged DMPG vesicle surface in order to test a recent suggestion (Riske et al., Chem. Phys. Lipids, 89 (1997) 31-44) that alterations in the surface potential accompanied apparent phase transitions observed by light scattering. The temperature dependence of the label partition between the lipid surface and the aqueous medium indicated an increase in the surface potential at the gel to liquid-crystal transition, supporting the previous suggestion. Results at the phase transition occurring at a higher temperature were less definitive. Although some change in the dCAT1 ESR spectra was observed, the interpretation of the phenomena is still rather unclear. DMPG surface potentials were estimated from the dCAT1 partition ratios (surface label moles/total label moles), using a simple two-sites model, where the electrostatic potential is zero everywhere but at the vesicle surface, and the interaction between the spin label and the membrane surface is chiefly electrostatic. The Gouy-Chapman-Stern model predicts surface potentials similar to those observed, although the measured decrease in the surface potential with ionic strength is somewhat steeper than that predicted by the model.

Role of soft and hard aggregates in the thermodynamics of lipid dispersions.

We study the thermodynamics of a two-dimensional polydisperse ideal gas model of different species of aggregates. We show that if these aggregates are distinguished not only by their sizes but also by their ability to display shape fluctuations, the system presents dominance of one or other species, depending on the temperature region. This result, which emerges solely from the statistics of the model in total absence of interaggregate interactions, describes well the observed temperature dependence of light scattering in dispersions of dimyristoyl phosphatidylglycerol, a negatively charged lipid.

Melanotropic peptides-lipid bilayer interaction. Comparison of the hormone alpha-MSH to a biologically more potent analog.

The interaction of the native peptide alpha-melanocyte stimulating hormone (alpha-MSH) and the biologically more active analog [Nle4, D-Phe7]-alpha-MSH(MSH-I) with lipid vesicles was studied by spin label electron spin resonance (ESR) spectroscopy and circular dichroism (CD). Using spin labels located at the membrane interface and at different depths along the acyl chain, it was shown that the binding of both peptides to the membrane induces tighter lipid packing at all the monitored positions. However, the effect of the analog on the spin label ESR parameters was much more evident, and suggested that it penetrates farthest into the lipid matrix than the native molecule. Lipid partition coefficients were calculated based on the effect the peptides cause on the ESR spectra of spin labels incorporated in the membrane. For the biologically more potent peptide, the partition coefficient was found to be about 4-times greater than that of the native hormone. For the same concentration of peptide bound to the membrane, MSH-I was found to cause a slightly greater effect on the membrane structure than alpha-MSH, in accord with its possible deeper penetration into the bilayer. CD spectra in aqueous solution and in the alpha-helix inducing solvent 2,2,2-trifluoroethanol showed that the two peptides have somewhat different structures in solution, though similar conformational changes occur in both peptides as a result of their interaction with negatively charged vesicles or micelles. The higher peptide-lipid association constant and the deeper penetration of the analog into lipid bilayers could be related to its greater activity and/or prolonged action.