Rhodopsin-phospholipid reconstitution by dialysis removal of octyl glucoside

Abstract

Recombinant membranes were prepared from phospholipid-free rhodopsin and egg phosphatidylcholine (PC) under a wide variety of conditions employing an octyl beta-D-glucoside (OG) dialysis procedure. Two bands were consistently observed after sucrose density centrifugation of these recombinants. The major band, which was protein rich, had a molar phospholipid:protein ratio that was in the range of 30:1 to 50:1, even when the molar phospholipid:protein ratio of the solubilized solution prior to OG removal was as high as 300:1. Similar results were obtained when dioleoyl-PC, 1-palmitoyl-2-oleoyl-PC, disk lipids, or diphytanoyl-PC was used instead of egg PC. These results can be explained in terms of a lower stability of the OG-phospholipid micelles relative to the OG-phospholipid-rhodopsin micelles. Of the phospholipids that were used in the OG dialysis procedure, only saturated dimyristoyl-PC produced a protein-rich recombinant band with a phospholipid:protein ratio close to that of the initial solubilized solution. In contrast to the results obtained by using OG, when solubilized disks supplemented with egg PC were reconstituted from sodium cholate or dodecyltrimethyl-ammonium bromide, the resulting recombinant membranes had initial and final phospholipid:protein ratios which were similar.