title: A fast way to track functional OmpF reconstitution in liposomes: Escherichia coli total lipid extract.
abstract: A major requirement to perform structural studies with membrane proteins is to define efficient reconstitution protocols that ensure a high incorporation degree and protein directionality and topology that mimics its in vivo conditions. For this kind of studies, protein reconstitution in membrane systems via a detergent-mediated pathway is usually successfully adopted because detergents are generally used in the initial isolation and purification of membrane proteins. This study reports OmpF reconstitution in preformed Escherichia coli liposomes followed by detection of its insertion by analyzing modifications on membrane structure by two different techniques: steady-state fluorescence anisotropy and dynamic light scattering. Another important issue is protein directionality. For OmpF, it is known that interaction with polyamines promotes channel blockage. In this work, the spermine-OmpF interaction was evaluated using surface plasmon resonance, and protein directionality was confirmed.
authors.affiliation: UCIBIO@Requimte, Faculdade de Ciências, Universidade do Porto, 4169-007 Porto, Portugal.
authors.forename: S C
authors.initials: SC
authors.lastname: Lopes
authors.affiliation: UCIBIO@Requimte, Faculdade de Ciências, Universidade do Porto, 4169-007 Porto, Portugal.
authors.forename: M
authors.initials: M
authors.lastname: Ferreira
authors.affiliation: UCIBIO@Requimte, Faculdade de Ciências, Universidade do Porto, 4169-007 Porto, Portugal.
authors.forename: C F
authors.initials: CF
authors.lastname: Sousa
authors.affiliation: UCIBIO@Requimte, Faculdade de Ciências, Universidade do Porto, 4169-007 Porto, Portugal. Electronic address: agsantos@fc.up.pt.
authors.forename: P
authors.initials: P
authors.lastname: Gameiro
mesh_terms: D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism; D005454:Fluorescence Polarization; D050356:Lipid Metabolism; D008055:Lipids / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry* / Q000378:metabolism; D018272:Porins / Q000737:chemistry* / Q000378:metabolism; D013096:Spermine / Q000378:metabolism
chemical_list: D008055:Lipids; D008081:Liposomes; C109237:OmpF protein; D018272:Porins; D013096:Spermine
keywords: Bacteria; Membrane protein; Protein reconstitution