Phosphatidylserine Allows Observation of the Calcium-Myristoyl Switch of Recoverin and Its Preferential Binding.

Recoverin undergoes a calcium-myristoyl switch during visual phototransduction. Indeed, calcium binding by recoverin results in the extrusion of its myristoyl group, which allows its membrane binding. However, the contribution of particular lipids and of specific amino acids of recoverin in its membrane binding has not yet been demonstrated. In the present work, the affinity of recoverin for the negatively charged phosphatidylserine has been clearly shown to be governed by a cluster of positively charged residues located in its N-terminal segment. Moreover, the calcium-myristoyl switch of recoverin was only observed upon binding onto monolayers of phosphatidylserine and not in the case of other anionic phospholipids. Fluorescence microscopy experiments with mixed lipid monolayers allowed confirmation of the specific binding of myristoylated recoverin to phosphatidylserine, whereas the extent of penetration of recoverin in phosphatidylserine monolayers was estimated by ellipsometry. A model has thus been proposed for the membrane binding of myristoylated recoverin in the presence of calcium.

Binding of methylene blue onto Langmuir monolayers representing cell membranes may explain its efficiency as photosensitizer in photodynamic therapy.

We provide evidence for the electrostatic interactions between the cationic photosensitizer methylene blue (MB) and cell membrane models represented by neat and mixed Langmuir monolayers of dioleylphosphatidylcholine (DOPC) and 1,1',2,2'-tetraoleoylcardiolipin (CL). From surface pressure measurements, MB was found to adsorb strongly and expand CL-containing monolayers, while it caused an apparent decreasing in molecular area on neat DOPC monolayer. The binding site of MB could be inferred from data with the surface-specific polarization-modulated infrared reflection-absorption spectroscopy (PM-IRRAS) technique, where changes induced by MB were observed in the vibrational modes of the phosphate groups of both CL and DOPC. The incorporation of MB also affected the carbonyl groups and the packing of the alkyl chains, thus indicating that MB binding site favors singlet oxygen generation close to the double bonds in the alkyl chains, an important requirement for photodynamic efficiency. Significantly, the data presented here demonstrate that MB may act in membranes composed by PCs, such as mammalian plasma membranes, and in those containing CL, as in bacterial and inner mitochondrial membranes.

Gel-assisted formation of giant unilamellar vesicles.

Giant unilamellar vesicles or GUVs are systems of choice as biomimetic models of cellular membranes. Although a variety of procedures exist for making single walled vesicles of tens of microns in size, the range of lipid compositions that can be used to grow GUVs by the conventional methods is quite limited, and many of the available methods involve energy input that can damage the lipids or other molecules present in the growing solution for embedment in the membrane or in the vesicle interior. Here, we show that a wide variety of lipids or lipid mixtures can grow into GUVs by swelling lipid precursor films on top of a dried polyvinyl alcohol gel surface in a swelling buffer that can contain diverse biorelevant molecules. Moreover, we show that the encapsulation potential of this method can be enhanced by combining polyvinyl alcohol-mediated growth with inverse-phase methods, which allow (bio)molecule complexation with the lipids.

Molecular organization of dengue fusion peptide in phospholipid monolayers revealed by tensiometry and vibrational spectroscopy.

The interaction of Dengue fusion peptide (FLAg) in selected lipid Langmuir monolayers was characterized with surface pressure-area isotherms and infrared spectroscopy to investigate the role of the membrane charge and molecular organization in the peptide-lipid binding. Surface pressure-area isotherms were employed to analyze the thermodynamic and mechanical properties of the FLAg-lipid monolayer, showing that charged lipid monolayers showed different peptide adsorption patterns for an optimized peptide concentration (maximum membrane adsorption). Polarization modulation infrared reflection-absorption spectroscopy pointed out that incorporating FLAg changed the dipole orientations for the lipid polar head groups, as confirmed in PG-containing monolayers. Also, FLAg reorients the lipid film when it interacts with the phosphate and choline groups. Finally, analysis of the 310-helix bands suggests that FLAg assumes a configuration as a hairpin, an essential premise for the beginning of the membrane fusion process.

Dengue fusion peptide in Langmuir monolayers: A binding parameter study.

Membrane fusion is known to be the primary mechanism of entry of flaviviruses into host cells. Several studies reported the investigation of the membrane fusion mechanism mediated by the fusion peptide, a component of the membrane protein surrounding the flaviviruses. In this study, we investigated the interaction of Dengue fusion peptide (FLAg) with Langmuir monolayers to uncover the role of membrane charges and organization in its membrane binding. Binding parameters of FLAg were obtained by measuring its adsorption onto Langmuir monolayers of different types of individual lipids, as well as their mixtures. Specific peptide binding was observed in the presence of charged lipid monolayers at different pHs, revealing that the lipid composition of the membrane modulates peptide interaction, and the preference of the peptide for negatively charged lipids.

Enhanced activity of horseradish peroxidase in Langmuir-Blodgett films of phospholipids.

The immobilization of enzymes in nanostructured films has potential applications, e.g. in biosensing, for which the activity may not only be preserved, but also enhanced if optimized conditions are identified. Optimization is not straightforward because several requirements must be fulfilled, including a suitable matrix and film-forming technique. In this study, we show that horseradish peroxidase (HRP) has its activity enhanced when immobilized in Langmuir-Blodgett (LB) films, in conjunction with dipalmitoylphosphatidylglycerol (DPPG). Incorporation of HRP into a DPPG monolayer at the air-water interface was demonstrated with compression isotherms, and Polarization-Modulation Infrared Reflection Absorption Spectroscopy (PM-IRRAS). From the PM-IRRAS data, we inferred that HRP was not denatured when adsorbed on a pre-formed, low pressure DPPG monolayer. A change in orientation was induced by the phospholipid matrix, with the amide C=O and NH groups from HRP being oriented perpendicular to the surface, parallel to the DPPG acyl chains, i.e. the alpha-helix was inserted into the monolayer. The mixed DPPG-HRP monolayer could be transferred onto solid supports, to which HRP activity was ca. 23% higher than in solution. The control of molecular architecture and choice of a suitable phospholipid matrix allowed HRP-containing LB films to be used in sensing peroxide.

Interaction of polysaccharide-protein complex from Agaricus blazei with Langmuir and Langmuir-Blodgett films of phospholipids.

The use of natural substances in health applications may be hampered by the difficulties in establishing the mechanisms of action, especially at molecular-level. The protein-polysaccharide complex extracted from the mushroom Agaricus blazei Murill, referred to as CAb, has been considered for treating various diseases with probable interaction with cell membranes. In this study, we investigate the interaction between CAb and a cell membrane model represented by a Langmuir monolayer of dimyristoyl phosphatidic acid (DMPA). CAb affects the structural properties of DMPA monolayers causing expansion and increasing compressibility. In addition, interaction with DMPA polar heads led to neutralization of the electrical double layer, yielding a zero surface potential at large areas per molecule. CAb remained at the interface even at high surface pressures, which allowed transfer of Langmuir-Blodgett (LB) films onto solid supports with the CAb-DMPA mixture. The mass transferred, according to quartz crystal microbalance (QCM) measurements, increased linearly with the number of deposited layers. With UV-vis absorption, fluorescence and FTIR spectroscopies, we confirmed that the LB films contain polysaccharides, proteins and DMPA. Therefore, the CAb biological action must be attributed not only to polysaccharides but also to proteins in the complex.