Which technique can be used to determine if a membrane protein is inside-out or right-side-out?
Determining orientation of membrane proteins after reconstitution into artificial vesicles essential for establishing vectorial transport competence. Proteoliposomes generated by removing detergent via dialysis may incorporate proteins randomly right-side-out exposing original extracellular domains outward or inside-out exposing cytoplasmic domains outward leading to mixed activity. Sidedness assay exploits membrane impermeability of antibodies one hundred fifty kilodaltons IgG unable to cross sealed bilayer. Intact vesicles incubated with monoclonal antibody against known extracellular epitope such as glycophorin N-terminus or Band 3 loop three undergo binding exclusively when epitope faces external medium detectable by secondary gold-conjugated antibody electron microscopy, flow cytometry, or after pelleting by immunoblotting pellet. Permeabilization with low concentration Triton X-100 zero point one percent exposes total epitopes providing total protein control. Alternative protease protection where extracellular trypsin cleaves only outward loops. Hydropathy plots predict number of spans not orientation in liposomes, SDS-PAGE separates subunits irrespective of sidedness, FRAP measures lateral mobility. Thus antibody labeling using impermeant probe provides definitive sidedness determination for inside-out versus right-side-out vesicles.
Ref: Rohde et al., Determination of Membrane Protein Orientation by Antibody Labeling, J Mol Biol Methods.