Classifications

• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
  • C08F8/00—Chemical modification by after-treatment
  • C08F8/30—Introducing nitrogen atoms or nitrogen-containing groups
  • C08F8/32—Introducing nitrogen atoms or nitrogen-containing groups by reaction with amines
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
  • C08F20/00—Homopolymers and copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride, ester, amide, imide or nitrile thereof
  • C08F20/02—Monocarboxylic acids having less than ten carbon atoms, Derivatives thereof
  • C08F20/52—Amides or imides
  • C08F20/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
  • C08F20/56—Acrylamide; Methacrylamide
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
  • C08F8/00—Chemical modification by after-treatment
  • C08F8/44—Preparation of metal salts or ammonium salts
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
  • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
  • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
  • C08F220/04—Acids; Metal salts or ammonium salts thereof
  • C08F220/06—Acrylic acid; Methacrylic acid; Metal salts or ammonium salts thereof

Definitions

• amphipols amphiphilic polymers
• amphipatic polymers are known in the state of the art for allowing the stabilization of membrane proteins in aqueous solution once they are extracted from the membranes via the use of detergents.
• Membrane proteins represent around 30% of the proteins encoded by the cell genome and are involved in many important cellular functions (signaling, secretion, migration, adhesion, homeostasis, energy production, etc.).
• the membrane proteins can be monomeric or oligomeric. By their integration into the membranes, they are strongly structured in helices a or sheets b for the transmembrane parts interacting with the alkyl chains of the membrane lipids.
• the surface of the extra-membrane domains of these proteins is mainly covered with amino acids with hydrophilic side chains while the surface of their transmembrane domain is covered with amino acids with hydrophobic chains; membrane proteins are therefore highly insoluble in water due to the hydrophobic effect which pushes them to aggregate in solution in order to minimize disturbances of the hydrogen bond network between water molecules.
• the detergents available on the market make it possible to dissolve the proteins but cause, as mentioned, their more or less rapid denaturation and inactivation in particular due to the dissociating power of the detergents; furthermore it is necessary to maintain these in solution at concentrations above their critical micellar concentration (CMC).
• CMC critical micellar concentration
• surfactants such as amphipols (WO 98/27434) make it possible to stabilize proteins but offer much weaker solubilizing properties.
• Amphipols are amphiphilic polymers defined as being capable of maintaining membrane proteins in their native, soluble conformation, in the form of small complexes (Zoonens & Popot, 2014). The most commonly used and studied amphipol is I ⁇ 8-35.
• SMAs for Styrene Maleic Acid copolymers, WO2011004158
• SMAs unlike amphipols which simply purify themselves at the end of their synthesis, always present impurities which pre-empt the subsequent use of proteins for biotechnological applications.
• Protein purification strategies and in particular membrane proteins, recombinant or not, are generally empirical and difficult to predict, depending in particular on the protein, the (micro) organism or the composition of the membrane; successive tests of different agents are generally carried out to solubilize and / or stabilize the membrane proteins, in order to identify the most effective agent under these conditions.
• the invention therefore aims to provide new compounds to allow the solubilization and stabilization of membrane proteins. More particularly, the invention relates to new amphipols, amphiphilic vinyl polymers having improved solubilization properties while allowing satisfactory stabilization of membrane proteins.
• the invention relates to amphiphilic vinyl polymers of formula I,
• M + is an alkali metal, preferably selected from Li + , Na + , K + ,
• Ri, R4, R5, Re are, each independently, the hydrogen atom or the methyl radical
• R2, R 6 , R9 are, each independently, a group selected from the hydrogen atom and the linear or branched (C1-C8) alkyls,
• R10 is a group selected from linear or branched (C1-C5) alkyls
• R3 and R 7 are, each independently, a group selected from: hydrogen, X then being a single bond
• poly or monounsaturated polycycles substituted or not substituted by one or more radicals selected from (C1-C8) linear, cyclic or branched alkyls, (C1-C8) linear or branched alkenyls, (C1-C8) linear or branched alkynyls, X then being a single bond or selected from among (C1-C8) linear or branched alkylene, (C2-C8) linear or branched alkenylenes and (C2-C8) linear or branched alkynylene,
• a phenyl substituted or not substituted by one or more radicals selected from (C1-C8) linear, cyclic or branched alkyls, (C1-C8) linear or branched alkenyls, (C1-C8) linear or branched alkynyls, X a single bond or being then selected from (C1-C8) linear or branched alkylene, (C2-C8) linear or branched alkenylenes and (C2-C8) linear or branched alkynylene, it being understood that when one of the groups R 3 or R 7 is a linear or branched alkyl of at least 6 carbon atoms, a linear or branched alkenyl of at least 6 carbon atoms, or a linear or branched alkynyl of at least 6 carbon atoms, or a d atom hydrogen, X then being a single bond, then the other group R 3 or R 7 is selected from: (C3-C10) cycloalkyls or
• poly or monounsaturated polycycles substituted or not substituted by one or more radicals selected from (C1-C8) linear, cyclic or branched alkyls, (C1-C8) linear or branched alkenyls, (C1 -C8) linear or branched alkynyls, X then being a single bond or selected from (C1-C8) linear or branched alkylene, (C2-C8) linear or branched alkenylene and (C2-C8) linear or branched alkynylene, and a phenyl, substituted or not by one or radicals selected from linear, cyclic or branched (C1-C8) alkyls, (C1-C8) linear or branched alkenyls, (C1-C8) linear or branched alkynyls, X then being a single bond or selected from (C1- C8) linear or branched alkylene, (C2-C8) linear or branched alkenylenes and the
• a phenyl substituted or not substituted by one or more radicals selected from (C1-C8) linear, cyclic or branched alkyls, (C1-C8) linear or branched alkenyls, (C1-C8) linear or branched alkynyls,
• X then being a single bond or selected from (C1-C8) linear or branched alkylene, (C2-C8) linear or branched alkenylenes and (C2-C8) linear or branched alkynylene.
• amphiphilic vinyl polymer of formula I is such that:
• R 3 and / or R 7 are (C6-C8) cycloalkyls as previously specified, or
• R 3 and / or R 7 are phenyl, preferably said phenyl is substituted in para by a methyl or ethyl group,
• X being a single bond, or selected from (C1-C8) linear or branched alkylene, (C2-C8) linear or branched alkenylenes and (C2-C8) linear or branched alkynylene.
• amphiphilic vinyl polymer of formula I is such that:
• R 3 and / or R 7 are (C6-C8) cycloalkyls as previously specified, X preferably being a single bond or CH2,
• R 3 and / or R 7 are phenyl, preferably said phenyl is substituted in para by a methyl or ethyl group, X being a single bond, or R 3 and / or R 7 are a styrenyl group. X being a simple bond.
• amphiphilic vinyl polymer of formula I is such that: the metal M is Na +,
• R 10 is an isopropyl group
• x is less than or equal to 75%
• - w and / or y is greater than or equal to 25%, w and / or y is between 25% and 50% and z is between 0% and 40%, preferably between 15% and 40%, or
• affinity tag grafting fluorescent probe or immunostimulatory molecule.
• the vinyl polymer of formula I is such that:
• Ri, R2, R4, R5, R6, Re, Rg are the hydrogen atom
• R3 and / or R7 is a ⁇ Ce-Cs) cycloalkyl and X is a single bond ,
• R10 is an isopropyl group
• x is equal to 35% or 50%
• - w is 0%, or 25%
• the vinyl polymer of formula I is selected from the compounds listed in Table I.
• the vinyl polymers of the invention are amphipols, that is to say that they are capable of keeping the membrane proteins soluble in their native conformation, in the form of small complexes.
• the invention therefore relates to the water-soluble complex formed by i) a membrane protein or else a mixture of membrane proteins and ii) at least one compound, amphiphilic vinyl polymer, as described above.
• the water-soluble complex thus formed is such that it also comprises lipid compounds.
• an object of the invention is the water-soluble complex formed by i) a membrane protein or else a mixture of membrane proteins and ii) at least one compound, amphiphilic vinyl polymer of the invention in which the membrane proteins are stabilized in solution aqueous.
• Another object of the invention is therefore a solution comprising such a complex.
• the invention also relates to a method for preparing an aqueous solution of membrane protein (s), said method comprising a solubilization and or stabilization step in which the protein fraction originating from a biological or synthetic membrane containing said membrane protein, or said mixture of membrane proteins, is placed in the presence of the vinyl polymer as described above.
• this process is such that: the membrane protein (s) are recombinant proteins expressed in prokaryotic or eukaryotic cells, and / or native proteins expressed in specialized membranes with high protein density, a step of solubilization of said membrane protein or of said mixture of membrane proteins is previously carried out by means of a detergent medium, a step of melting the protein fraction containing said membrane protein, or said mixture of membrane proteins, is previously carried out by means of vesicles of phospholipids or mixture of phospholipids, preferably 1, 2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), or the protein fraction solution containing said membrane protein is not a solution of membrane protein (s) (s) in a detergent medium in micellar form.
• DMPC 2-Dimyristoyl-sn-glycero-3-phosphocholine
• an object of the present invention is also the use of the complexes of the invention as reagents in reagent kits, and in particular a diagnostic kit comprising at least one complex of the invention as an immunological reagent.
• the complexes of the invention can be attached to a device making use of the activity of said membrane protein thus purified and solubilized by the amphipols of the invention for diagnostic or activity measurement purposes.
• Figure 2 Complexation of BR with amphipols according to the invention, before (black bars) or after centrifugation (gray bars).
• Figure 3 Thermal denaturation of BR in different environments.
• Figure 4 Solubilization of the purple membrane of Halobacterium salinarum before (A) or after (B) fusion with DMPC vesicles with I ⁇ 8-35, the SMAs or amphipols of the invention.
• Figure 5 Quantification of the BR extracted from the purple membrane, previously fused to the vesicles of DMPC, by I ⁇ 8-35, the SMAs, or of polymers of the invention (A) and analysis of the thermal stability of these complexes after 6 hours incubation at 50 ° C (B). The ratios of the absorbance values measured at the start and at the end of each experiment are reported.
• Figure 6 Extraction of the purple membrane.
• A Quantification of the solubilization (gray bars) and thermal stability (black bars) of the BR of the purple membrane, previously fused to the vesicles of DMPC, by I ⁇ 8-35, the SMAs, or of polymers of the invention each condition performed at least in triplicate values +/- SD;
• B Kinetics of thermal destabilization of complexes formed with SMAs or polymers 19 and 23 over 6 hours of incubation at 50 ° C. The absorbance values measured at 554 nm were normalized with those measured before incubation of the samples.
• Figure 7 Kinetics of solubilization of the protein YidC-GFP overexpressed in the plasma membrane of Escherichia coli in the presence of SMAs, polymers of the invention or DDM.
• A Photographs of the electrophoresis gels revealing the fluorescence of the GFP fused with the membrane protein YidC and
• B graphical representation of the quantification of the fluorescence from the image of the gels taking as reference the fluorescence measured in the presence of DDM after 1 hour of incubation.
• FIG. 8 Solubilization of the membranes rich in AChR of Torpedo marmorata with I ⁇ 8-35, the SMAs and amphipols of the invention.
• the samples corresponding to the supernatants (S) and pellets (C) of each condition were deposited on acrylamide gel, which after migration was stained with Coomassie blue.
• the arrows indicate the location of the bands corresponding to the AchR subunits and to the Torpedo marmorata Na7K + ATPase.
• the proteins present in the pellets are not solubilized, the proteins in the supernatant correspond to the solubilized proteins.
• the invention relates to new amphipols which allow the solubilization and stabilization of membrane proteins in an aqueous medium.
• the new amphipols of the invention have improved solubilization and / or stabilization properties compared to the reference polymers used to solubilize membrane proteins.
• the complexes thus formed with membrane proteins are particularly suitable for biotechnological applications and the study of these membrane proteins.
• amphipol designates vinyl polymers capable of keeping the membrane proteins soluble in their native conformation, in the form of small complexes.
• membrane protein is used in the singular or plural, it includes mixtures of membrane proteins or preparations comprising substantially only one type of protein.
• a membrane protein is a protein crossing on either side of the membrane, it then comprises at least one transmembrane domain or else a protein comprising at least one hydrophobic domain interacting with the membrane.
• the membrane protein can be monomeric or else oligomeric, associated or not with cofactors.
• a “stabilized” membrane protein corresponds to a solubilized membrane protein retaining a native conformation and, preferably, at least part of its biological activity, even if the latter may be temporarily blocked.
• specialized membranes includes, within the meaning of the invention, the membranes of eukaryotic organelles such as, for example, the crests of the mitochondria, the thylakoids of the chloroplasts, the endoplasmic or sarcoplasmic reticulum; "Specialized membrane” also includes membranes with high protein density comprising at least 50% of membrane proteins, integral or not, or even at least 60% or even at least 70% of membrane proteins, such as, for example, the purple membrane of the bacteria Halobacterium salinarum (which may include up to 75% bacteriorhodopsin), the rest comprising lipids or other molecules.
• biological membrane means, within the meaning of the invention, any lipid membrane extracted or originating from a cell or from an organism such as bacteria, fungus, eukaryotic cells etc.
• synthetic biological membrane designates any lipid membrane reconstituted, recomposed or modified with synthetic or naturally occurring lipids.
• a synthetic membrane may correspond to a lipid membrane extracted from an organism to which one or more synthetic lipids are added, a membrane made from synthetic lipids or a lipid membrane reconstituted from natural and or synthetic lipids intentionally mixed.
• protein fraction includes, within the meaning of the invention, the cell fraction containing the membrane protein sought or analyzed. Thus it may be a more or less clarified and / or purified cellular homogenate.
• protein fraction designates in particular a fraction of this homogenate such as the membrane fraction or else the fraction corresponding to a cell compartment.
• the invention therefore relates to an amphiphilic vinyl polymer of formula I:
• M + is an alkali metal, preferably selected from Li + , Na + , K + ,
• Ri , R4, R 5 , Re are, each independently, the hydrogen atom or the methyl radical
• R2, R 6 , R 9 are, each independently, a group selected from the hydrogen atom and the linear or branched (C1-C8) alkyls,
• R1 0 is a group selected from linear or branched (C1-C5) alkyls
• R 3 and R 7 are, each independently, a group selected from: hydrogen, X then being a single bond
• poly or monounsaturated polycycles substituted or not by one or more radicals selected from among (C1-C8) linear, cyclic or branched alkyls, (C1 -C8) linear or branched alkenyls, (C1 -C8) linear or branched alkynyls, X then being a single bond or selected from (C1-C8) linear or branched alkylene, (C2-C8) linear or branched alkenylenes and (C2-C8) linear or branched alkynylene, or
• a phenyl substituted or not by one or more radicals selected from linear (cyclic or branched) alkyls (C1-C8), linear or branched (C1-C8) alkenyls, (C1-C8) linear or branched alkynyls, X then being a single bond or selected from (C1-C8) linear or branched alkylene, (C2-C8) linear or branched alkenylene and (C2-C8) linear or branched alkynylene, it being understood that when one of the groups F3 ⁇ 4 or R 7 is a linear or branched alkyl of at least 6 carbon atoms, a linear or branched alkenyl of at least 6 carbon atoms, or a linear or branched alkynyl of at least 6 carbon atoms, or a hydrogen atom , X being a single bond, then, the other group F3 ⁇ 4 or R 7 is selected from: (C3-C10) cycloalkyls or
• poly or monounsaturated polycycles substituted or not substituted by one or more radicals selected from (C1-C8) linear, cyclic or branched alkyls, (C1-C8) linear or branched alkenyls, (C1-C8) linear or branched alkynyls, X then being a single bond or selected from (C1-C8) linear or branched alkylene, (C2-C8) linear or branched alkenylenes and (C2-C8) linear or branched alkynylene, or
• a phenyl substituted or not substituted by one or more radicals selected from (C1-C8) linear, cyclic or branched alkyls, (C1-C8) linear or branched alkenyls, (C1-C8) linear or branched alkynyls, X then being a single bond or selected from (C1-C8) linear or branched alkylene, (C2-C8) linear or branched alkenylene and (C2-C8) linear or branched alkynylene, w, x, y, z corresponding to the respective percentages of the units , x being between 20 and 90%,
• the polymer of formula I is such that: M + is an alkali metal, preferably selected from Li +, Na +, K +,
• Ri, R 4 , R 5 , Re are, each independently, the hydrogen atom or the methyl radical
• R 2 , R 6 , R 9 are, each independently, a group selected from the hydrogen atom and the linear or branched (C1-C8) alkyls, Rio is a group selected from (C1-C5) linear or branched alkyls,
• R 3 and / or R 7 are, each independently, a group selected from: (C3-C10) cycloalkyls or (C3-C10) (hetero) cycloalkyls, substituted or not by one or more radicals selected from (C1-C8 ) linear, cyclic or branched alkyls, the (C1-C8) linear or branched alkenyls and the (C1-C8) linear or branched alkynyls, X then being a single bond or selected from the (C1-C8) linear or branched alkylene, linear or branched (C2-C8) alkenylenes and linear or branched (C2-C8) alkynylenes,
• poly or monounsaturated polycycles substituted or not substituted by one or more radicals selected from (C1-C8) linear, cyclic or branched alkyls, (C1-C8) linear or branched alkenyls, (C1-C8) linear or branched alkynyls, X then being a single bond or selected from among (C1-C8) linear or branched alkylene, (C2-C8) linear or branched alkenylenes and (C2-C8) linear or branched alkynylene,
• a phenyl substituted or not substituted by one or more radicals selected from (C1-C8) linear, cyclic or branched alkyls, (C1-C8) linear or branched alkenyls, (C1-C8) linear or branched alkynyls, X a single bond or being then selected from (C1-C8) linear or branched alkylene, (C2-C8) linear or branched alkenylene and (C2-C8) linear or branched alkynylene, w, x, y, z corresponding to the respective percentages of the units , x being between 20 and 90%,
• the polymer of formula I is such that:
• M + is an alkali metal, preferably selected from Li + , Na + , K + ,
• Ri , F3 ⁇ 4, R 5 , Re are, each independently, the hydrogen atom or the methyl radical
• R 2 , R 6 , R 9 are, each independently, a group selected from the hydrogen atom and the linear or branched (C1-C8) alkyls,
• R 10 is a group selected from (C1-C5) linear or branched alkyls,
• R 3 and R 7 are, each independently, a group selected from: hydrogen, X then being a single bond
• a phenyl substituted or not substituted by one or more radicals selected from (C1-C8) linear, cyclic or branched alkyls, (C1-C8) linear or branched alkenyls, (C1-C8) linear or branched alkynyls, X then being a simple bond or selected from linear or branched (C1-C8) alkylene, (linear or branched C2-C8) and linear or branched (C2-C8) alkynylene, it being understood that when one of the groups F3 ⁇ 4 or R 7 is a linear or branched alkyl of at least 6 carbon atoms, a linear or branched alkenyl of at least 6 carbon atoms, or a linear or branched alkynyl of at least 6 carbon atoms, or a hydrogen atom, X being a single bond, then, the other group F3 ⁇ 4 or R 7 is selected from: (C3-C10) cycloalkyls or (C
• a phenyl substituted or not substituted by one or more radicals selected from (C1-C8) linear, cyclic or branched alkyls, (C1-C8) linear or branched alkenyls, (C1-C8) linear or branched alkynyls, X then being a single bond or selected from (C1-C8) linear or branched alkylene, (C2-C8) linear or branched alkenylene and (C2-C8) linear or branched alkynylene, w, x, y, z corresponding to the respective percentages of the units , x being between 20 and 90%,
• F3 ⁇ 4 and / or R 7 is a group selected from linear or branched alkyls of at least 6 carbon atoms, linear or branched alkenyls of at least 6 carbon atoms, or linear or branched alkynyls of at least 6 carbon atoms, X then being a single bond, then said alkyls, alkenyls, or alkynyls have less than 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, or less than 7 carbon atoms.
• the average molar mass of the amphipols according to the invention is less than 100,000 g. mol 1 , preferably between 2000 and 50,000 g. mol 1 .
• Embodiments of the invention are indicated below, which may or may not be taken in combination with another or between some of them.
• M + is Na + .
• y and / or w is greater than or equal to 25%.
• R 2 , R 6 , Rg are hydrogen.
• R 7 is a group selected from (C3-C10) cycloalkyls or (C3-C10) (hetero) cycloalkyls, substituted or not by one or more radicals selected from (C1-C8) linear, cyclic or branched alkyls, the (C1-C8) linear or branched alkenyls and the (C1-C8) linear or branched alkynyls, X then being a single bond or selected from among (C1-C8) alkylene linear or branched, the linear or branched (C2-C8) alkenylenes and the linear or branched (C2-C8) alkynylenes.
• X is a single bond and R 7 is a C6 cycloalkyl, preferably a C7 cycloalkyl, even more preferably a C8 cycloalkyl.
• R 7 is a C6 cycloalkyl, preferably a C7 cycloalkyl, even more preferably a C8 cycloalkyl.
• X is a linear or branched (C1-C8) alkylene, preferably methyl or ethyl.
• R 7 is a phenyl, substituted or not by one or more radicals selected from (C1-C8) linear, cyclic or branched alkyls ((C1-C8) linear alkenyls or branched, (C1-C8) linear or branched alkynyls, X then being a single bond or selected from (C1-C8) linear or branched alkylene, (C2-C8) linear or branched alkenylenes and (C2-C8) alkynylene linear or branched.
• X is a single bond and said phenyl is substituted for para.
• said phenyl is substituted for para by a linear C1-C8 alkyl, even more preferably, said linear alkyl in para position is selected from methyl or ethyl groups.
• R 7 is a phenyl substituted or not by one or more radicals selected from among (C1-C8) linear, cyclic or branched alkyls ((C1-C8) linear or branched alkenyls , (C1-C8) linear or branched alkynyls and X is selected from linear or branched (C1-C8) alkylene, preferably C1 alkylene or C2 alkylene.
• w and y are different from 0 and R 3 and R 7 are, independently of one another: the (C3-C10) cycloalkyls or (C3-C10) (hetero) cycloalkyls, substituted or not by one or more radicals selected from linear, cyclic or branched (C1-C8) alkyls, linear or branched (C1-C8) alkenyls and linear or branched (C1-C8) alkynyls, X then being a single bond or selected from linear or branched (C1-C8) alkylene, linear or branched (C2-C8) alkenylenes and linear or branched (C2-C8) alkynylenes, (C3-C10) cycloalkenyls or (C3 -C10) (hetero) cycloalkenyls, poly or monounsaturated, substituted or not by one or more radicals selected from among (C1-C8) linear,
• poly or monounsaturated polycycles substituted or not substituted by one or more radicals selected from (C1-C8) linear, cyclic or branched alkyls, (C1-C8) linear or branched alkenyls, (C1-C8) linear or branched alkynyls, X then being a single bond or selected from (C1-C8) linear or branched alkylene, (C2-C8) linear or branched alkenylenes and (C2-C8) linear or branched alkynylene, or
• R 3 and R 7 are then independently of each other: a C6 cycloalkyl, preferably a C7 cycloalkyl, moreover more preferred is a C8 cycloalkyl,
• a phenyl even more preferably substituted in para by a linear C1-C8 alkyl when it is monosubstituted, even more preferably, said linear alkyl in the para position is selected from methyl or ethyl groups, X then being a single bond, or
• R3 and / or R7 is a group selected from (C3-C10) cycloalkyls or (C3-C10) (hetero) cycloalkyls, substituted or not by one or more radicals selected from (C1-C8) linear, cyclic or branched alkyls, the (C1-C8) linear or branched alkenyls and the (C1-C8) linear or branched alkynyls, X then being a single bond or selected from among (C1-C8) alkylene linear or branched, preferably methyl or ethyl.
• R 3 and / or R 7 is phenyl, substituted or not by one or more radicals selected from (C1-C8) linear, cyclic or branched alkyls (C1-C8) linear or branched alkenyls, (C1-C8) linear or branched alkynyls, X then being a single bond or selected from linear or branched (C1-C8) alkylene, preferably methyl or ethyl.
• X is a single bond and said phenyl is substituted for para.
• said phenyl is substituted for para by a linear C1-C8 alkyl, even more preferably, said linear alkyl in para position is selected from methyl or ethyl groups.
• z is different from 0; preferably Rio is then an isopropyl group, even more preferably, R10 is an isopropyl group and, with regard to the units comprising R 3 and / or R7, X is selected from linear or branched (C1 -C8) alkylene groups, ( C2-C8) linear or branched alkenylenes and the (C2-C8) linear or branched alkynylenes, R3 and / or R7 being selected from: the (C3-C10) cycloalkyls or (C3-C10) (hetero) cycloalkyls, substituted or unsubstituted by one or more radicals selected from linear, cyclic or branched (C1-C8) alkyls, linear or branched (C1-C8) alkenyls and linear or branched (C1-C8) alkynyls,
• C3-C10 cycloalkenyls or (C3-C10) (hetero) cycloalkenyls, poly or monounsaturated, substituted or not by one or more radicals selected from among (C1-C8) linear, cyclic or branched alkyls, (C1- C8) linear or branched alkenyls, (C1-C8) linear or branched alkynyls,
• poly or monounsaturated polycycles substituted or not substituted by one or more radicals chosen from (C1-C8) linear, cyclic or branched alkyls, (C1-C8) linear or branched alkenyls, (C1-C8) linear or branched alkynyls, or a phenyl, substituted or not substituted by one or more radicals selected from (C1-C8) linear, cyclic or branched alkyls, (C1-C8) linear or branched alkenyls, (C1-C8) linear or branched alkynyls.
• amphiphilic vinyl polymer of formula I is chosen from the compounds listed in table I below, M + being an alkali metal, preferably selected from Li + , Na + , K + ; in a particular embodiment, M + is Na + .
• the percentages are specified with an accuracy of plus or minus 10%.
• a percentage for a motif is specified as being 50%, it should be understood that this motif may be present between 45% and 55% in the polymer of the invention.
• x is less than or equal to 75%.
• w and / or y is between 25% and 50% and z is between 0% and 40%, preferably between 15% and 40%.
• w + y is greater than or equal to 25%, preferably less than 50%.
• the polymers according to the invention are selected from the compounds of Table I with the proportions w, x, y and z as specified in Table II below.
• the compounds of the invention are new amphipols which are particularly capable of solubilizing and stabilizing membrane proteins, in particular with regard to other polymers such as I ⁇ 8-35 or SMA.
• the complexes formed with membrane proteins are of particular interest concerning the study of membrane proteins as well as for the biotechnological applications of these proteins.
• the stabilization of proteins in their native state and / or at least in part the preservation of their activity is essential for these applications; as demonstrated in the experimental part, the polymers of the invention allow the solubilization of membrane proteins and this stabilization.
• the solubilization can be carried out without detergent, which is particularly advantageous.
• membrane proteins known to be involved in pathologies, as being drug targets or capable of being immunogenic are particularly preferred.
• the possible applications for these complexes are therefore, for example: NMR in a liquid or solid medium, electron microscopy, diagnosis: the search for circulating antibodies, soluble or carried by lymphocytes, vaccination or production of antibodies: presentation of membrane proteins as immunogens, detection / use of the enzymatic activity of membrane proteins when these are enzymes , in pharmacology, measurement of the affinity of cellular receptors for molecules of pharmaceutical interest.
• the functionalized polymers of the invention are particularly advantageous.
• Those skilled in the art will be able to identify the functionalization means relevant to the intended use of the polymers of the invention.
• such means can be selected from those presented by Le Bon et al. (2014).
• an object of the invention is the complexes formed by the polymers of the invention as described in the present application and the membrane proteins.
• the complexes of the invention may be present in an aqueous solution, in lyophilized form or else immobilized in a matrix or on a surface via the functional groups grafted on the polymer of the invention.
• the protein: amphipol ratio is between 1: 0.1 and 1: 10, preferably between 1: 1 and 1: 7.5, even more preferably between 1: 1, 5 and 1: 5.
• this complex is therefore substantially devoid of detergent.
• the water-soluble complexes of the invention comprise lipids or a mixture of lipids. These can be useful for dilute the membrane proteins, as explained in the experimental part, or else come from the membrane of the organism or from the organelle from which the membrane protein is solubilized and stabilized within the complex.
• said lipid is 1, 2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC).
• the water-soluble complex can comprise all of the subunits of a multimeric protein or any cofactor associated with the membrane protein, as shown by the experiments on solubilization and stabilization of bacteriorhodopsin presented in the experimental part.
• the invention also relates to a process for the preparation of an aqueous solution of stabilized membrane protein (s) present in complexes with the polymers of the invention.
• the new amphipols of the invention have the capacity to solubilize membrane proteins without this preliminary solubilization step by the use of a detergent preparation.
• an object of the invention is the use of polymers of the invention to solubilize and / or stabilize the membrane proteins.
• the method for preparing a membrane protein solution or a mixture of membrane proteins, according to the invention does not include the step of dissolution by means of a detergent preparation , the solubilization being carried out using one of the polymers of the invention.
• X is a single bond and R 7 is a C6 cycloalkyl, preferably a C7 cycloalkyl, even more preferably a C8 cycloalkyl.
• w 0 and R 7 is a phenyl, substituted or not by one or more radicals selected from (C1-C8) linear, cyclic or branched alkyls, (C1-C8) linear or branched alkenyls, (C1-C8) linear alkynyls or branched, X then being a single bond or selected from among (C1-C8) linear or branched alkylene, (C2-C8) linear or branched alkenylenes and (C2-C8) linear or branched alkynylene.
• X is a single bond and said phenyl is substituted for para. Even more preferably, when it is monosubstituted, said phenyl is substituted for para by a linear C1-C8 alkyl, even more preferably, said linear alkyl in para position is selected from methyl or ethyl groups.
• the amphipols of the invention have polyacrylic acid as a precursor. Their synthesis is therefore very similar to that of other amphipols described in Gohon et al. (2006). Those skilled in the art will be able to adapt without particular difficulty the protocol for randomly grafting the groups specified on the polyacrylic acid precursor via the formation of amide bonds between a primary amine carried by said groups and a fraction of the carboxylate groups carried by the precursor.
• the reaction is carried out in / ⁇ / - methylpyrrolidone (NMP) in the presence of dicyclohexylcarbodiimide (DCC).
• NMP / ⁇ / - methylpyrrolidone
• DCC dicyclohexylcarbodiimide
• SERCAIa sarco / endoplasmic reticulum Ca2 + -ATPase 1 a
• SMA Styrene maleic acid copolymer, formula:
• SMA (2: 1) is made up of two thirds of styrene and one third of maleic acid.
• the SMA (3: 1) contains three quarters of styrene and one quarter of maleic acid.
• A8-35 Amphipol of formula:
• E. coli Escherichia coli
• GFP green fluorescent protein
• AChR acetylcholine receptor
• EDTA ethylenediaminetetraacetic acid
• Tris Tris (hydroxymethyl) aminomethane
• the experimental data below were obtained using membrane fractions and / or proteins to study the properties of the new amphipols of the invention. These examples are not limiting, in particular, the relative efficacies of the amphipols of the invention can vary as a function of the membrane fraction or of the membrane protein considered. However, the improved solubilization and / or stabilization properties of the amphipols of the invention vis-à-vis the reference polymers are of general application.
• Example 1 synthesis of the amphipols of the invention
• the amphipols of the invention have polyacrylic acid as a precursor. Their synthesis is therefore very similar to that of I ⁇ 8-35 described in Gohon et al. (2006). It consists in randomly grafting the groups onto the polyacrylic acid precursor via the formation of amide bonds between a primary amine carried by the groups to be grafted and a fraction of the carboxylate groups carried by the precursor. The reaction is carried out in / V-methylpyrrolidone (NMP) in the presence of dicyclohexylcarbodiimide (DCC). The grafting and obtaining of the different patterns can be done sequentially.
• NMP V-methylpyrrolidone
• DCC dicyclohexylcarbodiimide
• the grafting of the groups corresponding to a fraction of the carboxylate groups of the precursor takes place in a second step, in NMP in the presence of 1-hydroxybenzotriazole (HOBt) / DCC.
• HOBt 1-hydroxybenzotriazole
• the chemical composition of the polymer is determined by an NMR analysis of the proton and of the carbon, coupled with a pH-metric assay.
• reaction medium is filtered on sintered glass (porosity 4) and then 1.5 g of sodium methanolate (2 eq., 27.8 mmol) are added to the filtrate. Agitation is continued for approximately 15 minutes.
• the reaction medium is then poured into 260 ml (10 equivalent volumes) of purified and deionized water (Milli-Q ® ) and the pH is adjusted to 10 (pH paper) with sodium hydroxide (11 N).
• the solution is kept under vigorous stirring for around thirty minutes then filtered through a sinter (porosity 4).
• the filtrate obtained is added manually dropwise to 21 ml of 2N hydrochloric acid (3 eq., 42 mmol) with vigorous stirring.
• the addition is carried out via a syringe, the tip of which is connected to a millipore filter system (0.45 ⁇ m PES prefilter and 0.2 ⁇ m cellulose acetate filter in series).
• the suspension obtained is filtered through a frit (porosity 4) and the precipitate is dissolved in 50 ml of "Milli-Q ® " water after addition of 1.9 ml (1.5 eq ., 21 mmol) of sodium hydroxide 11 N.
• This precipitation / solubilization cycle is repeated four times, then the basic solution finally obtained is dialyzed (Spectrapore membrane with cutoff threshold at 8000 g. Mol 1 ) for 24 hours against a solution of soda at 4.5 ⁇ 10 3 N and then lyophilized.
• Example 2 analysis of the amphipol particles according to the invention by steric exclusion chromatography (SEC).
• the purple membrane was solubilized in OTG (see example 2), then the BR complexed with the amphipols at a BR / amphipol mass ratio of 1: 5.
• the column was equilibrated with 20 mM sodium phosphate pH 7.0, 100 mM NaCl.
• the number of carbon atoms between the amide bond and the cycloalkyl group of the amphipols according to the invention seems to influence the size and the homogeneity of the particles which they form.
• the addition of a carbon between the amide bond and the cycloalkyl ring results in more homogeneous particles having an elution profile comparable to that of amphipol A8-35.
• the presence of two carbon atoms results in the formation of larger and less homogeneous particles.
• Concerning the amphipols grafted with a phenyl group e.g.
• the elution profiles indicate that the particles formed by these polymers have a size greater than 40 kDa and are less homogeneous compared to GA8-35.
• the homogeneity is very clearly improved by the addition of an ethyl group (e.g. elution profile of compound 8) and results in particles of size more in relation to those of GA8-35
• the polymers of the invention therefore constitute customizable tools which have, in an aqueous medium, self-organizing properties in particles compatible with the processes for the purification of proteins in terms of homogeneity. These particles are comparable with those formed by amphipol A8-35 which is a benchmark in the community. 3.
• Example 3 study of the stabilizing effect.
• the stabilizing effect for the membrane proteins of the amphipols according to the invention was tested on two model membrane proteins, SERCAI a (sarco / endoplasmic reticulum Ca 2+ -ATPase 1a) and the bacteriorhodopsin of Halobacterium salinarum.
• SERCAI a is a calcium pump which requires the hydrolysis of an ATP molecule to perform its transport function.
• This protein belongs to the “P-type” ATPase family and its transmembrane segments adopt conformational changes of large amplitudes to allow the passage of calcium ions.
• the purple color of the BR is indicative of its native conformation.
• the BR covalently but reversibly binds the retinal (a chromophore) via the formation of a Schiff base.
• the protonation of this base causes the displacement of the absorption peak of the retinal from -380 nm (free form) to 570 nm or -555 nm (bound form) depending on whether the BR is embedded in the membrane or dissolved in aqueous solution.
• the absorbance at 554 nm is therefore indicative of the native conformation of the BR.
• the vesicles of the rabbit sarcoplasmic reticulum were prepared from muscle tissue according to the protocol described in Champeil et al. (1985). These membranes are very rich in SERCAI a.
• the ATPase activity of SERCAIa was measured indirectly by coupling several enzymatic reactions.
• the assay was carried out in the presence of pyruvate kinase (0.1 mg / ml) and lactate dehydrogenase (0.1 mg / ml) in order, on the one hand, to maintain constant the concentration of ATP in the sample and , on the other hand, to monitor ATP consumption by measuring the decrease in absorbance at 340 nm of NADH over time.
• the sample buffer contains 100 mM KC1, 1 mM Mg 2+ , 0.1 mM Ca 2+ , 50 mM Tes-Tris (pH 7.5 at 20 ° C) and 1-5 mM ATP.
• the reaction medium also contains 1 mM phosphoenolpyruvate and 0.15 mM NADH. Production of the purple membrane and preparation of BR / amphipol complexes
• the preparation of the BR / amphipol complexes takes place according to the following stages: i) A culture of Halobacterium salinarum was carried out and the membrane fraction corresponding to the purple membrane was purified according to the protocol already described in Oesterhelt & Stoeckenius (1971 ).
• the purple membrane was solubilized in 10 mM sodium phosphate buffer, 100 mM NaCl, 100 mM OTG, for a 48 hour incubation in a cold room with shaking and in the dark. The sample was centrifuged for 20 min. at 200,000 g at 6 ° C. The supernatant was diluted with OTG-free buffer to lower the detergent concentration from 100 mM to 18 mM.
• the concentration of BR was determined by measuring the absorbance at 554 nm. The amount of amphipol added was calculated based on the mass of BR present in the sample, corresponding to a BR / amphipol mass ratio of 1: 5. After 20 min of incubation at 4 ° C, polystyrene beads (Bio-Beads ® SM2, BioRad) were added so that their mass corresponds to 20 times that of the detergent present in the sample. After 2 h of incubation at 4 ° C with shaking, the beads were removed.
• BR / A8-35 or BR / A8-75 complexes positive controls
• BR complexes with the amphipols of the invention was carried out according to the protocol of replacement and elimination of the detergent by adsorption on polystyrene beads (Bio-Beads ® , BioRad).
• Bio-Beads ® were added to the mixture in a weight ratio Bio-Beads ® / OTG 20: 1 and incubated for 2 hours at 4 ° C.
• the absorbance at 554 nm of each sample was measured before and after centrifugation at 100,000 g for 20 min.
• the percentage of BR is calculated with reference to the absorbance measured in OTG before centrifugation.
• the ratio of the BR protein with the various solubilizing agents tested is 1: 5, in a 20 mM sodium phosphate buffer, pH 7.0, 100 mM NaCl.
• the samples were incubated at each temperature for 30 min and then cooled on ice before reading the absorbance at 554 nm.
• Concerning BR samples in detergent (OTG, Octylthioglucoside) one step of centrifugation was performed to remove the protein aggregates.
• the absorbance of the samples at 554 nm was measured with a UV-visible spectrophotometer taking as reference protein-free buffer. The absorbance values are normalized with respect to the absorbance measured at 4 ° C.
• the thermal stability of the BR complexed with I ⁇ 8-35, I ⁇ 8-75 or the amphipols of the invention was analyzed.
• the capacity of these polymers to maintain soluble BR in its native form was analyzed by measuring the absorbance of the supernatants at 554 nm.
• the influence of the charge of the amphipols of the invention was studied by increasing the grafting rate of the polymers with isopropyl groups, which has the effect of reducing the overall charge of the amphipol. Reducing the load (via a grafting rate of isopropyl groups up to 40%) thus obtained improves the rate of BR present in its native form in the samples after replacement of the detergent by amphipol in the presence of Bio-Beads ® (compare, eg, the absorbances for compounds 7 and 13). Conversely, a lower amount of BR is found in the supernatant when the charge density is reduced (compare eg compound 11 vs. compound 15), which could be due to aggregation of BR.
• amphipols of the invention grafted with cycloalkyl groups and / or phenyl groups up to 50% have a charge density equivalent to SMA (2: 1) or greater than SMA (3: 1).
• SMA charge density equivalent to SMA (2: 1) or greater than SMA (3: 1).
• all the amphipol of the invention tested make it possible to obtain more BR in its native form than the two SMAs. ( Figure 2).
• the denaturation of the protein takes place in a very narrow temperature range, as indicated by the slope of the sigmoid obtained, with a molecular fusion temperature (Tm) around at 63 ° C ( Figure 3A).
• Tm molecular fusion temperature
• Figure 3A The Tm of the protein in the OTG detergent is significantly lower ( ⁇ 47 ° C, Figure 3A), which indicates a lower stabilization of the protein in OTG.
• SMA 3: 1
• SMA (2: 1) of ⁇ 45 ° C and ⁇ 49 ° C respectively.
• the Tm obtained with the amphipols according to the invention are significantly higher than those in SMA and for some comparable or even greater than that of the BR / A8-35 complexes (eg compounds 4 and 3, with Tm ⁇ 57 ° C and ⁇ 66 ° C respectively, Figure 3B).
• the amphipols according to the invention are more effective than the SMAs and can be at least as effective as the reference amphipol A8-35, or even more effective as the results obtained concerning SERCA1 suggest. . 4.
• Example 4 evaluation of the solubilizing effect of the amphipols of the invention on membranes with high density of membrane proteins.
• GA8-35 is known to not effectively dissolve SERCA1 in sarcoplasmic reticulum vesicles (Champeil et al., 2000). Typically, membrane proteins are always solubilized using detergents, which are then exchanged with GA8-35 to stabilize them.
• SMA Styrene Maleic Acid copolymer
• SMALPs for SMA lipid particies lipid particles
• the stability of BR in SMA is relatively poor compared to that of the same protein complexed with I ⁇ 8-35.
• the solubilization properties of SMA, A8-35 and amphipols according to the invention were compared for different types of membranes more or less rich in membrane proteins (E. coli membrane, electrical organs of T. marmorata , thylakoids of C. reinhardtii, purple membrane of H. salinarum).
• the results obtained are specific to the membranes tested and illustrate the different properties of the different polymers.
• a weakly effective polymer for one of these model membranes could be quite effective for another type of membrane because of its specific protein and / or lipid composition.
• the purple membrane of Halobacterium salinarum is prepared according to the protocol described in Oesterhelt & Stoeckenius (1971).
• the membranes extracted from electrical organs of Torpedo marmorata naturally rich in acetylcholine receptor (AChR) are prepared according to the protocol described in Sobel et al. (1977).
• the thylakoid membranes of Chlamydomonas reinhardtti are prepared according to the protocol described in Pierre et al. (1995).
• the membranes of Escherichia coli overexpressing the YidC protein, fused in the C-terminal position to GFP, are prepared after mechanical lysis of the bacteria with a cell mill (Constant Systems). Cell debris and unbroken cells are removed by low speed centrifugation. The supernatant is recovered and the membranes are pelleted by centrifugation at high speed (100,000 g for one hour).
• the protocol for preparing the membranes of E. coli is described in Angius et al. (2018).
• DMPC vesicles (50 mg / ml) were formed by extrusion after 10 passes through a polycarbonate membrane (Whatman) calibrated with pores of 100 nm in diameter.
• the DMPC vesicles and the purple membrane are mixed so as to obtain a BR: DMPC mass ratio of 1: 5, then incubated for 30 min in a sonication bath according to the protocol already described by Knowles et al. (2009).
• the sample buffer contains 20 mM sodium phosphate pH 7.0, 100 mM NaCl.
• the OTG solubilization (100 mM) was carried out as described above.
• the mass ratio BR / SMA or amphipol is 1: 6.25 and the mass ratio BR / DPMC is 1: 5.
• the buffer used is 20 mM sodium phosphate, pH 7.0, 100 mM NaCl as mentioned above.
• the samples were incubated overnight at room temperature, with shaking, then centrifuged 20 min at 100,000 g, and the absorbance measurements carried out on the supernatants.
• the absorbances are normalized on the absorbance value measured before the ultracentrifugation step. Solubilization of the membranes of E. a recombinant membrane protein
• YidC is a membrane protein conserved during evolution which has 6 transmembrane domains in bacteria with gram negative staining.
• a recombinant form fused with GFP is used as a “reporter” protein for the solubilization of membranes for the study of the properties of the amphipols of the invention.
• the total membrane proteins present in the extracts of E membranes. coli are dosed using a colorimetric test. The protein concentration is adjusted to 2 mg / ml with 20 mM Tris buffer pH 8, 150 mM NaCl.
• the total protein / amphipol or SMA mass ratio is 1: 1.
• the total protein / DDM mass ratio is 1: 5.
• the buffer used under all conditions is composed of 20 mM Tris, pH 8.0, 150 mM NaCl.
• the samples are centrifuged for 10 min at 250,000 g.
• pellets separated from the supernatants, are resuspended in the initial volume of solubilization with 20 mM Tris buffer pH 8, 150 mM NaCl containing 5% SDS.
• Charging blue compatible with a measurement of fluorescence on acrylamide gel is added to these samples. It is a charge blue prepared according to the protocol described in Drew et al. (2008). The samples, corresponding to the supernatants and pellets of each condition and taken at the times indicated, are deposited on a 12% acrylamide gel.
• the electrophoresis is carried out in the presence of SDS in the migration buffer containing Tris-glycine pH 8.3.
• the proteins are migrated into the gel for 1 hour by applying a current of 100 V and 20 mA per gel.
• the ImageJ software makes it possible to integrate the areas of each band to quantify the fluorescence. The results are normalized with respect to the fluorescence obtained in detergent (DDM - 1 hour).
• AChR is an intrinsic membrane protein.
• the electrical components of Torscherdo Marmorata are particularly rich in this protein. These organs are therefore an important source and model for studying the acetylcholine receptor.
• the total membrane proteins present in the extracts of Torpedo marmorata membranes are assayed using a colorimetric test.
• the protein concentration is adjusted to 1 mg / ml with 5 mM NaP buffer pH 7.2, 100 mM NaCl, 1 mM EDTA.
• the total protein / amphipol or SMA mass ratio is 1: 5.
• the total protein / CHAPS mass ratio is 1: 4.
• the samples are incubated for one hour at 4 ° C, with shaking before being centrifuged for 20 min at 100,000 g.
• the pellets, separated from the supernatants, are resuspended in the initial volume of solubilization with 5 mM NaPi buffer pH 7.2, 100 mM NaCl, 1 mM EDTA.
• the samples corresponding to the supernatants and pellets of each condition, are mixed with charge blue prepared according to the protocol described in Laemmli (1970) before being deposited on a 10% acrylamide gel.
• the electrophoresis is carried out in the presence of SDS in the migration buffer containing Tris-glycine pH 8.3.
• the proteins are migrated into the gel for 1 hour by applying a current of 100 V and 20 mA per gel.
• each gel is colored with Coomassie blue and a photo of the gels, in white light, is taken.
• the purple membrane of Halobacterium salinarum comprises 75% of BR for 25% of lipids. Due to this unique composition, its solubilization by SMA is only possible when the membrane is previously fused with DPMC vesicles, in order to “dilute” the BR protein in lipids (Knowles et al., 2009; Orwick- Rydmark et al., 2012). Under these conditions, the amphipol of the invention are also capable of solubilizing BR, while GA8-35 remains ineffective (Figure 4B).
• amphipols of the invention comprising a grafting with a phenyl group
• the addition of an ethyl group on the aromatic site makes it possible to increase the solubilization properties (compare the percentage of BR solubilized for compounds 4, 6 and 8).
• the location of the ethyl group in para (position 4) or ortho (position 1) does not seem to matter, compounds 8 and 24 allowing a comparable solubilization (Figure 4B).
• amphipols of the invention having a grafting with a cycloalkyl are particularly effective with regard to the solubilization of BR.
• Compound 23 in particular is found to be as effective as SMA (2: 1) (compare the size of the pellets) but the amount of BR present in its native form in the supernatant is higher than that obtained with SMA (2: 1) or SMA (3: 1); compound 21 being significantly more effective than SMA (2: 1).
• the amphipol polymers of the invention exhibit better efficiency.
• extraction of the recombinant protein of interest as I ⁇ 8-35 and SMAs. Indeed, after two hours of incubation, in the presence of the amphipol polymers of the invention, the amount of protein extracted is approximately 80%, against approximately 35% in the presence of A8-35 or SMAs.
• the kinetics of extraction of the membrane protein of interest is faster (Figure 7).
• the amphipols of the invention are effective at low concentrations and allow faster extraction, which is particularly advantageous, in particular in the context of production of recombinant membrane proteins.
• This result indicates that the molecular architecture of the hydrophobic group can play an important role in the amphipols of the invention.
• amphipols of the invention are also particularly more effective in solubilizing the sodium potassium pump of the membranes of the electrical organs of T. marmorata than A8-50 or A8-35.
• amphipols of the invention also prove to be more effective in solubilizing AchR or the sodium potassium pump than A8-35, A8-50 and SMA 3-1 (data not shown) .
• the purple membrane was fused to DMPC vesicles according to the protocol described by Orwick-Rydmark et al. (2012) before initiating the solubilization step with the amphipols. After 24 hours of incubation at room temperature, the samples were centrifuged at 100,000X g for 20 min. The supernatants were separated from the pellet.
• the polymers of the invention tested make it possible to dissolve BR as effectively as SMA (3: 1) while stabilizing the protein at least as effectively as I ⁇ 8-35 (compare the absorbances for compounds 19 and 23 with the reference polymers, Figures 5 A and B and Figure 6). 6.
• the new amphipols of the present invention constitute particularly advantageous new polymers.
• the purification of membrane proteins requires solubilizing them while preserving their conformation and their stability, these two stages can seem contradictory, the solubilization implying in particular the use of surfactants with dissociative power which generally lead in the course of time to denaturation at least partial membrane proteins.
• the results show that the new amphipols of the invention have interesting solubilization properties for the purification of membrane proteins, while preserving the stability of these proteins and therefore their conformation and their biological activity, which is essential for their study or their use in biotechnological applications and not observed with the polymers of the prior art.
• the polymers of the invention are also effective at low concentrations, which is particularly advantageous for these applications.

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