Liposomal Reconstitution of Monotopic Integral Membrane Proteins

In spite of considerable progress in the methodology for reconstitution of membrane proteins into the liposomes, a successful reconstitution still appears to be more an art than a science. Reconstitution of membrane proteins into bilayers is required for establishing several aspects of the functions of membrane proteins and lipids and for elaborating models of naturally occurring membranes. Cyclooxygenase (COX)-1 and -2 (also prostaglandin endoperoxide H2 synthase, PGHS-1 and -2) belong to the class of monotopic membrane proteins. Membrane-binding domains of both COX-1 and -2 contain four short, consecutive, amphipathic α-helices (A, B, C, and D). Crystal structures of the COXs indicate that basic, hydrophobic, and aromatic residues in the membrane-binding domain are oriented away from the protein core and form a surface on the enzyme, which has been proposed to interact with the lipid bilayer (1). In this chapter, we describe a fast and efficient method for direct incorporation of COX-1 and -2 isozymes - as models for monotopic integral membrane proteins - into preformed liposomes containing fatty acids without loss of activity.