Insertion of glycophorin A, a transmembraneous protein, in lipid bilayers can be mediated by electropermeabilization

Transmembraneous back-insertion of a solubilized membrane protein, glycophorin A, has been obtained in 1,2 dipalmitoyl-sn-glycero-3-phosphocholine (Pam2GroPCho) cell-size-like liposomes by submitting the lipid/protein mixture to calibrated electric field pulses. Field conditions which are prone to trigger glycophorin insertion are similar to those which mediate lipid layer electropermeabilization. The efflux of calcein, trapped in the liposomes during their preparation, was observed only when field strength is higher than 1.3 kV/cm. Electroinsertion was detected only above the same critical field intensity. Calcein efflux as well as glycophorin insertion were increased by increasing field intensity, pulse duration and/or number of pulses. Experimental evidence of protein insertion was provided by physico-chemical as well as biochemical methods. Direct observation of the pulsed vesicles under the microscope revealed the insertion by means of immunofluorescence and fluorescence. Electroinsertion of fluorescent glycophorin A revealed that the inner bilayers were also labeled. The gel-to-liquid phase transition temperature of Pam2GroPCho decreased after insertion but its cooperativity was not affected. A narrow 31P-NMR peak was observed after electroinsertion showing that the polar headgroups of phospholipids had been altered. Analysis of trypsin-digested peptides revealed that the two trans-orientations of the protein across the external lipid layer were present after electroinsertion. Localized perturbation of the polar headgroup region of phospholipids, which supports the transient permeabilization of lipid layers, allows spontaneous transinsertion of glycophorin across the lipid bilayers.