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INTERACTION LIPIDE-PROTEINE WITH THE WATER-AIR INTERFACE

synopsis

I. INTRODUCTION 3 II. PRINCIPLE AND RECALLS 3 A. Principle 3 B. the plasmic membrane 3 1. Its organization and its components 3 a) Proteins 4 b) Lipids 5 c) Physical status of the membrane lipids 7 2. Roles and properties of membrane 7 III. EXPERIMENT AND EQUIPMENT 8 A. Equipment 8 B. Theory 9 1. Curve proportions 9 2. Kinetics of interaction 10 3. Curve of specificity 11 IV. APPLICATIONS 12 V. CONCLUSION 15

COUNT OF THE ILLUSTRATIONS

Figure 1: Diagrammatic representation of the plasmic membrane 4 Figure 2: Glycosphongolipides 5 Figure 3: The sphyngolipides 6 Figure 4: Cholesterol 7 Figure 5: Ferment LANGMUIR 8 Figure 6: Injection of solution 9 Figure 7: Curve proportions 10 Figure 8: 10 Figure 9: Kinetics of interaction 11 Figure 10: Curve of Specificity 11 Figure 11: Diagrammatic representation of virus VH1 12 Figure 12: Fusion of the VIH1 on the level of a microdomain glycolipidic 14

I. INTRODUCTION

The interactions lipids/proteins with the air/water interface are studied within the Laboratory of Biochemistry and Physicochemistry of the Biological Membranes by the team of the pr. Jacques FANTINI. Their goal is to study the way in which the components of the biological membrane interact between them and with the external elements. These interactions can be carried out:  is between lipids  is between lipids and proteins

II. PRINCIPLE AND RECALLS

A. Principle The use of monomolecular films makes it possible to study the interaction of proteins and lipids with a monomolecular lipidic film. The integration of a molecule in full-course of lipids induces a variation of the pressure of surface to the interface water/air. B. the plasmic membrane 1. Its organization and its components

The membrane has two roles which seem “incompatible”:  To isolate an entity alive from its environment  To communicate selectively with this environment (exchange of matter and information). The membrane is essential to the course of the biological processes because it maintains the differences essential between the contents of the compartments which it delimits, and their environment. It thus forms borders whose crossing is highly controlled. The biological membrane is double-layered phospholipidique, average thickness 7,5 Nm, in which are inserted, in an asymmetrical way, of the macromolecular buildings of proteinic and/or glycoproteic nature. Thus, one can observe two types of slopes:  extracellular which carries sugars associated with proteins or lipids  intracellular, almost ever glycosylées.

The various elements constituting this structure are not bound by covalence, but by various types of weak connections (force of Van Der Waals, bridges hydrogen).

FIGURE 1: REPRESENTATION SHEMATIQUE OF MEMBRANE PLASMIQUE

a) Proteins

According to their origin, the membranes contain from 40 to protein 70% distributed in the double-layered lipidic one, these are the proteins which will give a function specific to the membrane concerned. According to the importance of their hydrophobic part, one distinguishes:  integral or intrinsic proteins They are included in the lipidic matrix. These last can be isolated only when the membrane fitting is destroyed using detergents. The two layers of the membrane can contain:  Is fields different from an intrinsic protein,  Is different proteins, which increases their asymmetry.

 extrinsic proteins These proteins make it possible to establish interactions with proteins anchored beforehand to the membranes. Their role is thus to maintain proteins in the vicinity of the membrane.  glycoprotéines and glycolipides The plasmic membrane also comprises compounds polysaccharidic (2 to 10%), in the form of glycoprotéines and of glycolipides. These compounds polysaccharidic are very absorbent and are positioned on the external face of the plasmic membrane, still accentuating its asymmetry. These osidic compositions play a big role in the phenomena of communication and intercellular recognition.

b) Lipids

The membrane lipids are of small size and amphipathic (a hydrophobic pole and an absorbent pole). One can thus distinguish:  the hydrophobic pole which is represented by the two tails with long chains of fatty-acid, directed inside the structure  the pole absorbent represented by the head of the lipids, and directed towards the outside of the structure.

The principal types of lipids which one meets in the membranes are:  of the glycérophospholipides which are majority membrane lipids with 90%. They are built on the basis of glycerol.

FIGURE 2: GLYCOSPHINGOLIPIDES (GALL CER)

 of the sphingolipides which are formed starting from sphingosine which is a carbonaceous chain with 18 carbons. A sphingosine and an fatty-acid form a céramide. The sphingolipides are a group of lipids including:  a hydrophobic part (céramide)  an absorbent grouping. The skeleton of all the sphingolipides is a céramide which results from the condensation of a serine with an fatty-acid, palmitic acid generally, followed by a acylation (addition of an fatty-acid). These fatty-acids are usually saturated and comprise from 14 to 30 carbon atoms. Those can present or not a characteristic to the level of the carbon atom located in  of the grouping CO.  If OH is in  carboxyl, then it acts of a hydroxylated fatty-acid .  If there is no OH, then it acts of a nonhydroxylated fatty-acid.

The sphingolipides can be separate in 2 classes:  If, to the céramide one adds a phosphorylcholine, one obtains a sphingomyelin then.  If, to the céramide one adds a sugar, one obtains a glycosphingolipide then.

Sphingomyelin is the most widespread sphingolipide in the cellular membranes. It is present in the membranes of all the cellular organoids, but 90% are localized in the external layer of the plasmic membrane (about the extracellular middle). The contents out of cholesterol and sphingomyelin of the membranes are narrowly correlated. These two molecules are present in the same membrane microdomains.

FIGURE 3: SPHINGOLIPIDES

 cholesterol (only in the animals) which intervenes in the fluidity and the mechanical stability of the membrane.

FIGURE 4: CHOLESTEROL

c) Physical status of the membrane lipids

 Concept of melting point (Tm)

The melting point is the temperature of transition freezing-liquid. The existence of a heterogeneity of lipids in the membrane presenting of Tm different results in the presence of microdomains. In the membrane, the presence of glycophospholipides induces a fluid lipidic phase (for a rate of 90%) and that of sphingolipides, a solid phase. For example, for oil and butter when one wants to mix them, it is necessary to heat to exceed the Tm of butter and to obtain the liquid phase. Thus, the sphingolipides will gather in called lipidic microdomains “raft” which are not disorganized by the action of detergents. This property differentiates them from the remainder from double-layered lipidic. The microdomains of glycosphingolipides could be highlighted at the level of the plasmic membrane thanks to their insolubility in the detergent triton-X100 at 4°C (Brown and Rose, 1992). In fact, the lipids, which are in a fluid state are generally built-in mixed micelles under the action of the detergent, whereas the lipids which are in a gelled state or ordered liquid are not solubilized. Certain membrane fields correspond to areas specialized in term of composition out of proteins 2. Roles and properties of the membrane

The double-layered phospholipidique one presents a double role:  solvent of hydrophobic proteins  barrier of permeability (with respect to polar substances and of substances charged). With regard to its supporting role a point is to be retained. Indeed, because of its hydrophobic central part, the double-layered lipidic one has a low permeability to the absorbent substances like the polar ions, water and molecules. Thus, the ions can passively cross the membrane only on the specialized protein level: protein-channels. In the same way, they actively cross the membrane only on the level of the pumps or the conveyers. This organization allows the regulation of the passage of the ions, passage focused on the protein level whose opening (protein-channels) or operation (pumps - conveyers) is narrowly controlled.

This double-layered phospholipidique is impermeable with the water-soluble compounds; it is fluid because each element can turn on its axis and diffuse quickly in the side direction. This degree of fluidity will depend on the composition of double-layered (membrane model of the “fluid mosaic” by Singer and Nicholson), but also according to the conditions of the medium and in particular of the temperature (fluidity tends to increase with the temperature). So that a membrane effectively achieves its activity and its functions, it is necessary that it has an optimal fluidity. The fluidity of the lipidoproteic matrix is at the origin of the capacity of fusion of the membranes and the homogenization of the components of the structure thus reconstituted. These properties are fundamental for the exchanges will intra and extracellular. This capacity of fusion is due to the universality of the membrane structure and nonspecific nature of the hydrophobic interactions which maintain the integrity of the structure and which guides its constitution.

III. EXPERIMENT AND EQUIPMENT A. Equipment

We use a tensiometer to measure the surface tension of full-course lipidic after insertion of a solution containing of proteins. The operation of the tensiometer rests on the principle of the “balance of Langmuir”.

FIGURE 5: FERMENT LANGMUIR We have a Teflon plate with a multitude of tanks (15 well by plates). In the tanks will be placed first of all sterile water. One will place the platinum needle at a depth of two millimetres in order to make the zero and before the formation of the lipidic moncouche which will be formed around the needle. We will inject then the deposit of lipids in order to obtain the full-course one with the interface.

FIGURE 6: INJECTION OF THE SOLUTION

Once the formed monomolecular film we inject a solution containing of the proteins which will come to intercalate itself between the lipids of full-course what will cause an increase in the pressure of surface. The needle will record the variations of the state of compaction of film according to time, after the injection of the solution containing of proteins. The needle is connected to the tensiometer which will bring back the evolutions of the tension. All information is sent to a computer equipped with the software which can treat the data. We will visualize the increase in the pressure of surface via a curve plotted by the computer.

B. Theory 1. Curve proportions

Once the monomolecular film created, a “curve amount” will be realized in order to know the peptide concentration to be injected. Several concentrations are tested, them  max is then deferred on a graph according to the peptide concentration. The concentration has to inject selected corresponds to a  max on the “plate” of the curve.

FIGURE 7: CURVE PROPORTIONS 2. Kinetics of interaction

A curve of kinetics of interaction is carried out with the pressure of selected insertion of the lipidic layer (10 mN/m). Shortly after the injection, one observes an increase in the pressure of the molecules on the surface of monomolecular film, at the moment when peptide forms part of this last. A  max is observed. The operation is repeated for the various pressures of insertion (from 10 to 30 mN/m).

FIGURE 8:

FIGURE 9: KINETICS Of INTERACTION 3. Curve of specificity

The  max are deferred according to the various initial pressures. One prolongs the curve of tendency formed by the points. The point where the curve cuts the x-axis characterizes the critical pressure of insertion. If this critical pressure of insertion exceeds a certain value (approximately 20-25 mN/m), it is said that the interaction is specific between peptide and the lipid, peptide injected has enough affinity to intercalate itself in the middle of the lipids forming monomolecular film.

FIGURE 10: CURVE OF SPECIFICITY

IV. APPLICATIONS

The virus of the human immunodéficience (HIV) belongs to the family of the retroviruses which are the only viruses diploides because, indeed, their genome consists of two molecules of identical ARN of positive polarities, connected by a pseudo-pairing of ends 5 '. The viral particles are spherical forms made up of a nucléocapside surrounded by a roughcast external envelope of spicules. The penetration of the nucléocapside inside the target cell is ensured by its adhesion the plasmic membrane, on the level of specific receiving sites, followed fusion of the viral envelope with the plasmic membrane of the target cell. The HIV gathers two distinct types:  HIV 1  HIV 2

The interaction of the VIH1 with the plasmic membrane of the target cells implies two glycoprotéines viral envelope:  Glycoprotéines external of surface ensuring the recognition of the cellular receivers (gp120)  Glycoprotéines transmembrane ensuring fusion itself (gp41)

FIGURE 11: REPRESENTATION SHEMATIQUE OF VIRUS VH1

The fixing of the VIH-1 on the membrane of the target cell implies major conformational changes on the level of the glycoprotéine of surface, the gp120. The gp120 sets on the receiver CD4, receiver proteinic localized in the rafts, on the surface of the target cell what involves the first conformational change. There is then appearance of the V3 loop, which until was now partially masked, representing the hypervariable field gp120 playing a crucial role in fusion virus-cell. The area of the gp120 including/understanding the V3 loop interacts with a cofactor of fusion of the target cell, which allows the interaction of the peptide of fusion with the plasmic membrane of this cell. The peptide of fusion is protected in the hydrophobic zone consisted specific folding up from the gp120. When this one is fixed on the coreceptor, the second conformational change is set up and there is eradication of the gp41 of this zone. The peptide of fusion of the gp41 is then confronted with the aqueous medium and fits in the plasmic membrane of the target cell to minimize this energy constraint. The nature of the cofactor can vary but the two principal ones are CXCR4 and CCR5 which are proteins with seven membrane fields. This complex mechanism of fusion requires the regrouping of various membrane proteins on the level of the site of fusion, called raft. At the origin, only receiver CD4 is associated with the raft. When the virus is fixed at it, it is embarked on the raft then drift in the membrane until reaching the functional coreceptor. However if the gp120 is absent, CXCR4 does not bind physically, because this one induces the reorganization of the raft to deliver the CD4-gp120 complex to the functional corécepteurs. FIGURE 12: FUSION OF THE VIH1 ON THE LEVEL OF A MICRODOMAIN GLYCOLIPIDIQUE

V. CONCLUSION

The plasmic membrane, of share its heterogeneity of structure (composition, fluidity) is the seat many biological processes. These processes can be of nature various but that which relate to the interactions between lipids and lipids/proteins on the level of the membranes, holds a relatively important role. This property indeed made it possible to the researchers to include/understand how was held certain biological phenomena which can take a worrying importance nowadays (like that of the interaction of the HIV or the prion with the membranes). Consequently thus, these studies enable us to hope to control, with more precision, the various stages which will possibly be able to lead to the appearance of a disease, and to plan to find via complementary research, of the means which will be able either to slow down it, or to stop it.

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