Orientation of transmembrane polypeptides as revealed by antibody quenching of fluorescence

We describe here a new method, based on fluorescent techniques, for the determination of the orientation of membrane protein molecules present in vesicles. The method consists of: (a) attachment of a fluorescein derivative to sugar residues of glycoproteins and glycolipids in the cell membrane, and (b) the use of anti-fluorescein antibody, a highly efficient quencher of fluorescein fluorescence, for the quantitative evaluation of sidedness of transmembrane orientation of protein molecules in vesicles. Since antibody molecules do not permeate membranes, quenching is limited exclusively to sites exposed at the external surface of the vesicles. Addition of antibody to a fluorescently-labeled cell suspension results in a full and immediate quenching of the fluorescent signal. The method is highly sensitive (pM protein concentration), rapid and readily applicable to various vesicle preparations. With this method we assessed the orientation of vesicles derived from red blood cell membranes (ghosts) in isotonic medium and followed their inversion from right-side-out to inside-out orientation upon incubation in alkaline, low ionic strength medium.