Interaction of cysteine and its derivatives with monolayers of dipalmitoylphosphatidylcholine.

Molecular interactions between l-cysteine (Cys) and its ester derivatives (Cysx); l-cysteine ethyl ester (CE), l-cysteine methyl ester (CM) and N-acetyl l-cysteine (NAC) with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) monolayers were investigated using Langmuir film balance technique. The effect of charge on monolayers made of cysteine and three ester derivatives with DPPC was investigated by working with un-buffered and buffered subphases. Also, the effects of cysteine derivatives interaction with DPPC monolayers were studied measuring the change in the surface tension upon aminoacid injection in the subphase whilst keeping lipid molecular density and lateral packing controlled. Cysteine and its ester derivatives showed interfacial activity reducing the air/water surface tension (πi) by 4 mN m-1. However, ester derivatives were able to insert into preformed DPPC monolayers at much higher surface pressures (Δπ), indicating a preferential interaction of Cysx with DPPC. The results indicate that, although the different derivatives of cysteine presented low surface activity, they were able to favourably interact with DPPC monolayers. Also, compression isotherms experiments in binary mixtures indicate that the more surface active compounds stabilized the gel phase of DPPC. The charge on cysteine and its derivatives did not increase the observed effects.

The case for class II bacteriocins: A biophysical approach using "suicide probes" in receptor-free hosts to study their mechanism of action.

Class II bacteriocins are unmodified membrane-active peptides that act over a narrow spectrum of target bacteria. They bind a specific receptor protein on the membrane to form a pore, leading to membrane permeabilization and cell death. However, little is known about the molecular events triggering the pore formation after the bacteriocin recognizes the receptor. It is not clear yet if the pore is the same receptor forced into an open conformation or if the pore results from the bacteriocin insertion and oligomeric assembly in the lipid bilayer. In order to reveal which model is more suitable to explain the toxicity mechanism, in this work we use chimeric peptides, resulting from the fusion of the bitopic membrane protein EtpM with different class II bacteriocins: enterocin CRL35, pediocin PA-1 and microcin V. E. coli strains lacking the specific receptors for these bacteriocins were chosen as expression hosts. As these constructs display a lethal effect when they are heterologously expressed, they are called "suicide probes". The results suggest that, indeed, the specific receptor would act as a docking molecule more than as a structural piece of the pore, as long as the bacteriocin is somehow anchored to the membrane. These set of chimeric peptides also represent an in vivo system that allows to study the interaction of the bacteriocins with real bacterial membranes, instead of model membranes. Hence, the effects of these suicide probes in membrane fluidity and transmembrane potential were also assessed, using fluorescence spectroscopy. The data show that the different suicide probes are able to increase phospholipid order and depolarize the membranes of receptor-free bacterial cells.

Hexagonal phase with ordered acyl chains formed by a short chain asymmetric ceramide.

Ceramides constitute a group of lipids with usually high melting temperature that also favor negative curvature in membranes when mixed with other lipids. The short chain C10:0 ceramide is an asymmetric lipid which consists of an 18 carbon sphingosine base N-acylated with decanoic acid. According to high sensitivity differential scanning calorimetry, it shows a minor exothermic peak at 61°C and a main endothermic transition at 75°C. By small angle X-ray scattering and polarized light microscopy we found that, at temperatures below the main transition, the fully hydrated lipid dispersions are arranged in a tridimensional structure corresponding to an inverted hexagonal phase. Infrared spectroscopy and wide angle X-ray diffraction indicated that the acyl chains of ceramides exhibit a relatively high order in the hexagonal phase. As far as we know, this is the first report of a lipid hexagonal phase having highly ordered acyl chains. Molecular asymmetry due to the different length of the sphingosine and the N-acyl chains of C10:0 ceramide may explain why this novel phase is formed.

The antimicrobial peptide microcin J25 stabilizes the gel phase of bacterial model membranes.

The bacterial membrane interaction of the antimicrobial peptide microcin J25 was studied with the probe-free techniques Langmuir monolayers and infrared spectroscopy. Membrane model systems composed by phosphatidylethanolamine:phosphatidylglycerol 7:3, which mimic the cytoplasmic membrane of Gram negative bacteria, were used in both monolayer and bilayer approaches. The peptide reduced the transition surface pressure of the expanded-to-condensed lipid monolayer states, as well as increased the gel-to-liquid crystalline transition temperature in bilayers, indicating a stabilization of membrane ordered state. In addition, a reduction of the surface pressure at which condensed domains appeared was observed upon mixed monolayers compression after microcin J25 adsorption. The results indicate a favorable interaction of microcin J25 with bacterial membrane model systems. Also, the effects on the ordered phases stabilization are discussed in terms of the biological effects observed in membranes of sensitive cells.