Nicole Van Mau

Domain Formation in Models of the Renal Brush Border Membrane Outer Leaflet

The plasma membrane outer leaflet plays a key role in determining the existence of rafts and detergent-resistant membrane domains. Monolayers with lipid composition mimicking that of the outer leaflet of renal brush border membranes (BBM) have been deposited on mica and studied by atomic force microscopy. Sphingomyelin (SM) and palmitoyloleoyl phosphatidylcholine (POPC) mixtures, at molar ratios varying from 2:1 to 4:1, were phase-separated into liquid condensed (LC) SM-enriched phase and liquid expanded (LE) POPC-enriched phase. The LC phase accounted for 33 and 58% of the monolayers surface for 2:1 and 4:1 mixtures, respectively. Addition of 20-50 mol % cholesterol (Chl) to the SM/POPC (3:1) mixtures induced marked changes in the topology of monolayers. Whereas Chl promoted the connection between SM domains at 20 mol %, increasing Chl concentration progressively reduced the size of domains and the height differences between the phases. Lateral heterogeneity was, however, still present at 33 mol % Chl. The results indicate that the lipid composition of the outer leaflet is most likely responsible for the BBM thermotropic transition properties. They also strongly suggest that the common maneuver that consists of depleting membrane cholesterol to suppress rafts does not abolish the lateral heterogeneity of BBM membranes.

Detection of Peptide-Lipid Interactions in Mixed Monolayers, Using Isotherms, Atomic Force Microscopy, and Fourier Transform Infrared Analyses

To improve the understanding of the membrane uptake of an amphipathic and positively charged vector peptide, we studied the interactions of this peptide with different phospholipids, the nature of whose polar headgroups and physical states were varied. Three lipids were considered: dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), and dioleoylphosphatidylglycerol (DOPG). The approach was carried out by three complementary methods: compression isotherms of monolayers and atomic force microscopy observations associated with Fourier transform infrared investigations. From analysis of the compression isotherms, it was concluded that the peptide interacts with all lipids and with an expansion of the mean molecular area, implying that both components form nonideal mixtures. The expansion was larger in the case of DOPG than for DPPC and DPPG because of an alpha to beta conformational transition with an increase in the peptide molar fraction. Atomic force microscopy observations showed that the presence of small amounts of peptide led to the appearance of bowl-like particles and that an increase in the peptide amounts generated the formation of filaments. In the case of DOPG, filaments were found at higher peptide molar fractions than already observed for DOPC because of the presence of negatively charged lipid headgroups.

Protein structural changes induced by their uptake at interfaces

For insertion into lipidic media, most hydrosoluble proteins must cross the lipid-water interface and thus undergo conformational transitions. According to their chemical sequences these transitions may be restricted to changes involving only the tertiary structure, while for other proteins this environment modification will induce drastic changes such as the unfolding of large domains. The structural transitions are mainly governed by the presence of hydrophobic domains and/or by the existence of induced amphipathic properties.

Ionic pores formed by cyclic peptides

It is shown that 2 cyclic tetrapeptides, namely tentoxin and HC toxin, are able to induce the formation of transmembrane ionic channels, although a carrier mechanism could be expected on the basis of their chemical structure (presence of proline or N-methylated residues). Since other cyclic peptides but of larger size, i.e., tyrocidines, gramicidin S (decapeptides) and an octapeptide with a sequence similar to that of HC toxin, are also able to form pores, it appears that this property can be extended to a large number of cyclic peptides. A pore structure based on aggregates is proposed.

SPECIFIC INTERACTION BETWEEN ANIONIC PHOSPHOLIPIDS AND MILK BOVINE COMPONENT PP3 AND ITS 119-135 C-TERMINAL FRAGMENT

Abstract The behaviour of component PP3, a bovine milk protein with emulsifying properties, was investigated at the air–water interface and in a lipidic environment using the monolayer technique. The amphipathic 119–135 C-terminal fragment of PP3 was also tested since we proposed, on the basis of structural analysis, that this region is probably responsible for the surface-active properties of the protein. This hypothesis was confirmed by the tensiometric measurements at the air–water interface in which the addition of the C-terminal peptide increased the surface pressure with a similar amplitude as the whole protein. Penetration measurements into lipidic monolayers indicated that the insertion of component PP3 and its C-terminal peptide is the highest with anionic phospholipids in a gel state. Moreover, the electrostatic attractions provided by anionic phospholipids are essential for the peptide interaction. We also showed by Fourier transform infrared spectra study, that the peptide displays a β-type conformational state in aqueous solution and in the presence of solvant or anionic phospholipid (DPPG). In contrast, the protein adopts in aqueous solution an α helical conformation which remains the dominant conformational state in the presence of DPPG although the apparition of β-structure is detected.

Single channels and surface potential of linear gramicidins.

The single channel data for 4 different linear gramicidins containing either 4 Trp, 4 Phe, 4 Tyr or TyrBzl have been analyzed on the basis of 3 barriers-2 sites model. They form 2 families which differ by their single channel behavior and thus different energy profiles of the channel. A relationship between the surface potential and the entry barrier is proposed.

Synthesis of a template-associated peptide designed as a transmembrane ion channel former.

We describe the design and the Fmoc/tBu solid phase synthesis of a 20 residue long peptide containing five regularly distributed lysines. Cyclization of this peptide was achieved using BOP as coupling agent. After side‐chain deprotection, all the basic residues were iodoacetylated and then allowed to react either with a C‐terminal free COOH peptide or with peptides bearing a cysteamide group. The final pentameric templates were identified by mass and amino acid analysis which gave data compatible with the expected values. Copyright

Primary Amphipathic Shuttle Peptides: Structural Requirements and Interactions with Model Membranes

Two families of primary amphipathic peptides issued from the association of a nuclear localization sequence (NLS) with either a signal (SP) or fusion peptide (FP) were shown to be efficient carriers of drugs or nucleic acids [1,2]. In order to define the minimal structural requirements that maintain the vector properties, the identification of the membrane crossing process was a crucial step. Here, we focus on the uptake of the vectors by model membranes and examine the influence of the hydrophobic sequence, and thus of its conformation, on the penetration into phospholipid monolayers of various physical states and polar headgroups.

THE ADSORPTION OF ENDOTHELIN 1 TO PHOSPHOLIPIDS IS GOVERNED BY ELECTROSTATIC INTERACTIONS. A MONOLAYER AND FLUORESCENCE STUDY

Abstract In order to elucidate the influence on the lipidic environment on the recognition process of its membrane associated receptor, the interactions of the vasoconstrictor peptide endothelin 1 with various phospholipids have been investigated using different lipidic model membranes: monolayers at constant surface pressure, vesicles and micelles. A monolayer study of ET1 adsorbed onto the water surface has shown that the C-terminus of the peptide points towards the aqueous phase. Penetration measurements into lipidic monolayers indicate that ET1 adsorbs to phospholipids with an orientation similar to that of the air–water interface and fluorescence measurements are in agreement with such an orientation of the peptide. This adsorption is selective for neutral phospholipids and indicates that the nature of the phospholipid headgroups is of major importance for the approach of the membrane associated receptor.