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AssigneesUniversite Claude Bernard Lyon I, Centre National de la Recherche Scientifique (CNRS), Institut National Des Sciences Appliquees De Lyon, Ecole Superieure De Chimie Physique Electronique De Lyon
InventorsAgnes GIRARD-EGROT, Ofelia MANITI, Samuel REBAUD
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The invention concerns an assembly consisting of a substrate on which at least one lipid bilayer is attached by means of a peptide, referred to as the tethering peptide, which is itself linked to the substrate, characterised in that the tethering peptide has a C-terminal end constituted by at least four consecutive histidines and in that the lipid bilayer comprises a portion of lipids having a chelating headgroup enclosing a metal cation providing the link with the tethering peptide as a result of metal-chelate interactions between the metal cation and at least a portion of the histidines located at the C-terminal position of the tethering peptide; and the method for preparing same and the associated detection methods.
1- Assembly consisting of a substrate on which at least one lipid bilayer is attached by means of a peptide, referred to as the tethering peptide, which is itself linked to the substrate, characterized in that the tethering peptide has a C-terminal end consisting of at least four consecutive histidines and in that the lipid bilayer comprises a portion of lipids having a chelating polar headgroup enclosing a metal cation providing the link with the tethering peptide as a result of metal-chelate interactions between the metal cation and at least a portion of the histidines constituting the C-terminal end of the tethering peptide.
2- Assembly according to claim 1 characterized in that the metal cation is a nickel cation, a gadolinium cation or a copper cation.
3- Assembly according to claim 1 characterized in that the metal cation is a nickel cation and the chelating polar headgroup is nitrilotriacetic acid.
4- Assembly according to claim 1 characterized in that the portion of lipids having a chelating polar headgroup enclosing a metal cation represents from 0.5 to 5 mol %, preferably from 1 to 2 mol %, of the totality of the lipids forming the lipid bilayer.
5- Assembly according to claim 1 characterized in that the tethering peptide is attached covalently to the substrate, preferably by its N-terminal end.
6- Assembly according to claim 1 characterized in that the substrate is made of gold.
7- Assembly according to claim 6 characterized in that the tethering peptide comprises a cysteine at its N-terminal end establishing an S—Au bond with the gold substrate.
8- Assembly according to claim 1 characterized in that the C-terminal end of the tethering peptide consists of four, five or six consecutive histidines.
9- Assembly according to claim 1 characterized in that the link with the tethering peptide is provided as a result of metal-chelate interactions established between the metal cation and two of the histidines located in the C-terminal portion of the tethering peptide.
10- Assembly according to claim 1 characterized in that the composition of the lipid bilayer is selected so as to mimic a biological membrane, and notably a cell membrane.
11- Assembly according to claim 1 characterized in that the lipid bilayer is fluid and continuous.
12- Assembly according to claim 1 characterized in that a membrane protein, preferably an integral membrane protein, is inserted into the lipid bilayer.
13- Assembly according to claim 1 characterized in that the substrate has several zones on which a lipid bilayer is attached by means of a peptide, referred to as the tethering peptide, said tethering peptide having a C-terminal end consisting of at least four consecutive histidines and the lipid bilayer comprising a portion of lipids having a chelating polar headgroup which encloses a metal cation and provides the link with the tethering peptide as a result of metal-chelate interactions between the metal cation and at least a portion of the histidines located in the C-terminal portion of the tethering peptide.
14- Method for preparing an assembly according to claim 1 characterized in that it comprises the following successive steps:
15- Preparation method according to claim 14 characterized in that the lipid bilayer is obtained by fusion of unilamellar liposomes having an average diameter ranging between 30 and 500 nm.
16- Preparation method according to claim 14 characterized in that the liposomes are proteoliposomes.
17- Preparation method according to claim 14 characterized in that fusion is obtained by means of a fusogenic peptide, preferably of SEQ ID NO 2.
18- Method of detection by surface plasmon resonance imaging using an assembly according to claim 1 wherein the substrate is made of gold.