INTERACTION OF PARTIALLY UNFOLDED FORMS OF TORPEDO ACETYLCHOLINESTERASE WITH LIPOSOMES

A water-soluble dimeric form of acetylcholinesterase from electric org an tissue of Torpedo californica was obtained by solubilization with p hosphatidylinositol-specific phospholipase C of the glycophosphatidyli nositol anchored species, followed by purification by affinity chromat ography. The water-soluble species, in its catalytically active native conformation, did not interact with unilamellar vesicles of dimyristo ylphosphatidylcholine. We previously showed that either chemical modif ication or exposure to low concentrations of guanidine hydrochloride c onverted the native enzyme to compact, partially unfolded species with the physicochemical characteristics of the molten globule state. In t he present study, it was shown that such molten globule species, wheth er produced by mild denaturation or by chemical modification, interact ed efficiently with small unilamellar vesicles. Binding was not accomp anied by significant vesicle fusion, but transient leakage occurred at the rime of binding. The bound acetylcholinesterase reduced the trans ition temperature of the vesicles slightly, and NMR data suggested tha t it interacted primarily with the head-group region of the bilayer. T he effects of tryptic digestion of the bound acetylcholinesterase were monitored by gel electrophoresis under denaturing conditions. It was found that a single polypeptide, of mass similar to 5 kDa, remained as sociated with the vesicles. Sequencing revealed that this is a tryptic peptide corresponding to the sequence Glu 268-Lys 315. This polypepti de contains the longest hydrophobic sequence in the protein, Leu 274-M et 308, as identified on the basis of hydropathy plots. Inspection of the three-dimensional structure of acetylcholinesterase reveals that t his hydrophobic sequence is largely devoid of tertiary structure and i s localized primarily on the surface of the protein. It is suggested t hat this hydrophobic sequence is aligned parallel to the surface of th e vesicle membrane, with nonpolar residues undergoing shallow penetrat ion into the bilayer.