Characterization of membrane protein reconstitution in LUVs of different lipid composition by fluorescence anisotropy.
A major requirement to perform structural studies with membrane proteins is not only to define efficient reconstitution protocols, that assure a high incorporation degree in preformed liposomes, but also a protein directionality and topology that mimics its in vivo conditions. For this kind of studies, protein reconstitution in membranes systems via a detergent-mediated pathway is usually successfully adopted, since detergents are generally used in the initial isolation and purification of membrane proteins. In this study we report the reconstitution of OmpF in preformed DMPC and E. coli liposomes using two different techniques for detergent removal: (1) exclusion chromatography and (2) incubation with detergent-adsorbing beads. The incorporation degree was determined by bicinchoninic acid assay and fluorescence anisotropy was used to determine OmpF effect on the structural order of membrane lipids. These results show that protein insertion in membranes depends both on the technique used to remove detergent and on the lipids used to prepare the liposomes. Furthermore, it is possible to state that although the insertion is directly related to the size distributions of proteoliposomes, it could be efficiently recognized by steady-state fluorescence anisotropy. This technique, more popular among cell biologists, can be a very practical and straightforward alternative to DLS to confirm membrane protein insertion.
Neves
Patrícia
P
Requimte, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, 4169-007 Porto, Portugal.
Lopes
Silvia C D N
SC
Sousa
Isabel
I
Garcia
Sónia
S
Eaton
Peter
P
Gameiro
Paula
P
D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D002850:Chromatography, Gel / Q000379:methods; D003902:Detergents; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D005454:Fluorescence Polarization / Q000379:methods; D008027:Light; D050356:Lipid Metabolism; D008055:Lipids / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry* / Q000378:metabolism; D008563:Membrane Lipids / Q000378:metabolism; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008566:Membranes / Q000378:metabolism*; D018625:Microscopy, Atomic Force / Q000379:methods; D011506:Proteins / Q000378:metabolism*; D011510:Proteolipids; D015203:Reproducibility of Results; D012542:Scattering, Radiation
D001425:Bacterial Outer Membrane Proteins; D003902:Detergents; D029968:Escherichia coli Proteins; D008055:Lipids; D008081:Liposomes; D008563:Membrane Lipids; D008565:Membrane Proteins; D011506:Proteins; D011510:Proteolipids; C020485:proteoliposomes