TOWARD ELECTROPHORETIC SEPARATION of MEMBRANE PROTEINS in SUPPORTED n-BILAYERS

Frédéric Harb (1) , Bernard Tinland (1)

1 CINaM - Centre Interdisciplinaire de Nanoscience de Marseille

Membrane proteins are key constituents of the proteome of cells but are poorly characterized, mainly because they are difficult to solubilize. Proteome analysis involves separating proteins as a preliminary step towards their characterization. Currently, the most common method is "solubilizing" them with sophisticated detergent and lipid mixtures for later separation via, for instance, sodium dodecyl sulfate polyacrylamide gel electrophoresis. However, this later step induces loss of 3D structure (denaturation). Migration in a medium that mimics the cell membrane should therefore be more appropriate. Here, we present a successful electrophoretic separation of a mixture first of two and then of three different membrane objects in supported n-bilayers. These "objects" are composed of membrane proteins Sulfide Quinone Reductase and α-Hemolysin. Sulfide Quinone Reductase forms an object from three monomers together and self-inserts into the upper leaflet. α-Hemolysin inserts as a spanning heptamer into a bilayer or can build stable dimers of α-Hemolysin heptamers under certain conditions. By appropriately adjusting pH it proved possible to move them in different ways. This work holds promise for separating membrane proteins without losing their 3D structure, thus their bioactivity, within a lipidic environment that is closer to physiological conditions and for building drug/diagnostic platforms.

Mots clés: Electrophoresis; Membrane; Mobility; Proteins; Separation

Domaines: Biophysique; Génomique, Transcriptomique et Protéomique [q-bio.GN]

Frédéric Harb, Bernard Tinland. TOWARD ELECTROPHORETIC SEPARATION of MEMBRANE PROTEINS in SUPPORTED n-BILAYERS. 2020. ⟨hal-02482576v4⟩