Jean-Paul Chauvet

Behavior of a GPI-anchored protein in phospholipid monolayers at the air–water interface

The interaction between alkaline phosphatase (AP), a glycosylphosphatidylinositol (GPI)-anchored protein (AP-GPI), and phospholipids was monitored using Langmuir isotherms and PM-IRRAS spectroscopy. AP-GPI was injected under C16 phospholipid monolayers with either a neutral polar head (1,2-dipalmitoyl-sn-glycero-3-phosphocholine monohydrate (DPPC)) or an anionic polar head (1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS)). The increase in molecular area due to the injection of protein depended on the surface pressure and the type of phospholipid. At all surface pressures, it was highest in the case of DPPS monolayers. The surface elasticity coefficient E, determined from the pi-A diagrams, allowed to deduct that the AP-GPI-phospholipid mixtures presented a molecular arrangement less condensed than the corresponding pure phospholipid films. PM-IRRAS spectra suggested different protein-lipid interactions as a function of the nature of the lipids. AP-GPI modified the organization of the DPPS deuterated chains whereas AP-GPI affected only the polar group of DPPC at low surface pressure (8 mN/m). Different protein hydration layers between the DPPC and DPPS monolayers were suggested to explain these results. PM-IRRAS spectra of AP-GPI in the presence of lipids showed a shape similar to those collected for pure AP-GPI, indicating a similar orientation of AP-GPI in the presence or absence of phospholipids, where the active sites of the enzyme are turned outside of the membrane.

Penetration of a GPI-anchored protein into phospholipid monolayers spread at the air/water interface

Abstract Mammalian alkaline phosphatases (AP) belong to glycosylphosphatidyl inositol (GPI) anchored proteins family, which are localised and clustered on the outer layer of the plasma membranes forming microdomains. Using Langmuir film and polarisation modulation infrared reflection absorption spectroscopy (PMIRRAS) techniques, the penetration process of the protein into a phospholipid monolayer have been studied at the air–buffer interface. The penetration of AP-GPI in distearoylphosphatidylcholine monolayers (DSPC) induces a more important surface pressure increase than in dioleoylphosphatidylcholine (DOPC) monolayer. However, the exclusion surface pressure rather similar for both lipids, 20.5 and 22 mN m−1 for, respectively, DSPC and DOPC, indicates that the AP-GPI cannot, in similar conditions, insert by itself into bilayer membranes of either biological or mimetic origin. PMIRRAS suggests that the pure acyl chains perdeuterated DSPC (d70-DSPC) interact with Mg2+ present into the buffer. AP-GPI inserts progressively into the d70-DSPC monolayer changing the environment of phospholipid molecules. Amide I band exhibits α helix and β-sheets components with a predominance of the α helix. The shapes, intensities and positions of the amide I and II bands suggest for the α helix an orientation perpendicular to the interface after a period of molecular reorganisation.

Control of Immobilization and Hybridization on DNA Chips by Fluorescence Spectroscopy

A new methodology for the analysis of DNA polymorphisms has been developed using specific oligonucleotide strand arrays bound to a solid silicon support recovered by a thin layer of silica. Arrays of directly synthesized oligonucleotides covalently fixed on Si/SiO2 wafers have been designed at the macroscopic scale. Using suitable nucleotide-labeled units, the fluorescence emission technique has been used as an experimental control of the molecular network bound to the support and as a method for analyzing the hybridizing abilities of the corresponding oligonucleotide array. Fluorescein has allowed us to control the molecular density of the DNA strand resulting from a complete synthetic growing process. A specific protocol using both complementary and noncomplementary units labeled with two probes, Cy3 and Cy5, was used to distinguish clearly nucleotide units fixed on the array either as hybridized sequences or by the unavoidable adsorption process. The present performance of this fluorescence detection procedure will now be used with a scanning fluorescence device to perform the analysis at the microscopic scale.

IgG1-glycolipidic LB films obtained by vertical deposition of an interfacial film formed through proteo-liposome spreading at the air/water interface

Abstract This study focuses on the formation of an immunoglobulin–glycolipid interfacial film through the disintegration of proteo-glycolipid liposomes at the air/buffer interface. The IgG-glycolipid liposomes were formed by mechanical dispersion and were spread on the buffer surface. The time-dependent increase in surface pressure following the liposome spreading was attributed to the formation of a surface film due to the interfacial reorganisation of the proteo-glycolipid vesicles. The presence of immunoglobulin in the glycolipid matrix was evidenced by the modification of the elastic properties of the interfacial film during the film compression. The transfer efficiency of the immunoglobulin with the glycolipid matrix by vertical Langmuir–Blodgett (LB) deposition procedure has been characterised through ATR–FTIR and fluorescence spectroscopies. Observations performed with Epi-fluorescence microscopy gives a direct evidence of the presence of immunoglobulin in the LB film.

Synthesis and interfacial properties of amphiphatic cryptophanes

The new amphiphatic cryptophanes M5 and M6 were synthesized from their precursor M4. They present interesting properties due to their long chain substituents on one of the cyclotriveratrylene moieties. M4 was synthesized from a thio-cyclotriveratrylene platform obtained from thio-vanillin, using the template method developed for the preparation of dissymmetrical cryptophanes. The tris-hexanol (M5) or tris-hexadecyl (M6) substituted cryptophanes exhibited enhanced amphiphatic properties, which were investigated through the π–A isotherms of the interfacial films obtained at the air–water interface (Langmuir monolayers). The corresponding surface elasticity coefficients of the films were determined and their stability analyzed at different surface pressures. Stable molecular films of M5 were obtained after a first compression step, suggesting a rearrangement of the molecules at the interface. This behavior matched with a reduction of the molecular area consistent with the formation of bilayers of M5 molecules. Cryptophane M6 formed stable and reversible Langmuir monolayers at higher surface pressure (πmax = 27 mN m−1). This was attributed to interactions between the long alkyl chain substituents of the cryptophane, which favor the organization of the molecules on the water subphase. At higher pressure both compounds form aggregates irreversibly.

In Situ and Ex Situ Fabrication of DNA Chips by Microdeposition

DNA chips are devices associating the specific recognition properties of two DNA single strands through hybridisation process with the performance of microtechnology. It is expected that the use of microtechnology allows to obtain reproducible massively parallel analysis at a low cost [1].