Elaboration de dérivés du pullulane à amphiphilie contrôlée : Application à l'extraction de protéines membranaires intégrales

Elaboration of amphiphilic pullulan derivatives : Application to membrane proteins extraction

• Auteur / Autrice : Caroline Duval
• Direction : Guy Muller
• Type : Thèse de doctorat
• Discipline(s) : Chimie. Polymères fonctionnels
• Date : Soutenance en 2002
• Etablissement(s) : Rouen

Mots clés

Mots clés contrôlés

Protéines -- Dénaturation (chimie)

Polysaccharides

Surfactants

Protéines membranaires

Pullulane

Composés amphiphiles

Résumé

Les protéines membranaires intégrales très hydrophobes étant insolubles dans l'eau, l'emploi d'agents tensioactifs est nécessaire à leur purification et leur caractérisation en solution aqueuse. Parmi les tensioactifs récemment développés, des polymères amphiphiles synthétiques, les amphipols, présentent des potentialités intéressantes. Leur substitution par des biopolymères amphiphiles à propriétés similaires semblant très attractive, nous avons cherché à mettre au point des polysaccharides hydrophobiquement modifiés, capables d'extraire et de solubiliser des protéines membranaires dans des conditions non dénaturantes. Le polysaccharide choisi est le pullulane: hydrosoluble, flexible, sa modification chimique est bien maîtrisée. . .

Study of integral membrane proteins is of great importance for the understanding of cellular mechanisms, such as photosynthesis, respiration, or bacterial resistance to antibiotics. Those highly hydrophobic proteins are water insoluble; surfactants are thus necessary for their handling in aqueous solutions. Among surfactants recently developed for this use, amphiphilic polymers called amphipols display interesting potential. Amphiphilic biopolymers with similar properties would be particularly attractive. Therefore, we tried to develop hydrophobically-modified polysaccharides able to extract integral membrane proteins and to make them soluble, under non-denaturing conditions. Pullulan is a water-soluble, flexible polysaccharide. Its chemical modification is well controlled. A set of amphiphilic pullulan derivatives with moderate molar mass ( '' 30 000 g. Mol-1) was prepared. Carboxymethyl groups were first introduced (0,2 to 1,2 per anhydroglucose unit) to favor water-solubility of amphiphilic derivatives. Large extents (up to 48%) of alkyl (C8 to C12) and 3-phenylpropyl hydrophobic chains were then grafted through amide or ester linkages. Amphiphilic carboxymethylpullulans display surface-active properties. Hydrophobic microdomain formation was evidenced in aqueous solution. According to the samples studied, various aggregation phenomena were observed. Some amphiphilic carboxymethylpullulans are actually able to extract membrane proteins from their lipid surrounding. Comparison of their capacities led to identify C4?18C10d (pullulan with 18% decyl groups and 91% ungrafted carboxymethyl groups) as the most efficient sample. The effect of this amphiphilic biopolymer on protein activity is similar to that of a mild detergent