by Cys residues provided a protein that under nonreducing conditions was fully oxidized. The far-UV CD spectra of this mutant in the reduced and oxidized states indicated that their secondary structures were identical to the structure of the wild type recombinant apoLp-III, which contains no Cys residues. Near-UV CD studies confirmed the formation of a disulfide bond and the absence of structural perturbations. The lipid binding activity of the reduced mutant, as determined by its ability to form discoidal lipoproteins, was nearly identical to that of the wild type protein. Contrarily, the disulfide form of the mutant was not able to form discoidal lipoproteins with liposomes of either dimirystoylphosphatidylcholine or dimyristoylphosphatidylglycerol. It is concluded that the separation of the helices 1 and 5 constitutes one of the key steps along the complex pathway for the formation of the final apolipoprotein lipid-bound state. It is inferred that the conformational flexibility of helices 1 and 5 is a key property of apoLp-III, allowing the exposure of hydrophobic protein regions and the interaction of the hydrophobic faces of the amphipathic ␣-helices with the lipoprotein lipid surface.
Apolipophorin-III (apoLp-III)1 is an exchangeable apolipoprotein found in the hemolymph of many insect species in a lipid-free state or bound to the major insect lipoprotein, lipophorin. (1, 2). The structure of apoLp-III from two different insect species has been solved. The structures of Locusta migratoria and Manduca sexta apoLp-III were determined by x-ray crystallography (3) and NMR spectroscopy (4), respectively. The structure of apoLp-III is described as an elongated bundle of five amphipathic ␣-helices, where the nonpolar faces of the helices are oriented toward the protein core. A similar structure, but consisting of a four-helix bundle, is present in the N-terminal domain of human apoE (5). M. sexta and L. migratoria apoLp-III share a large number of physical-chemical properties with the apolipoproteins from humans and other vertebrates (1, 6, 7). Among the common features of exchangeable apolipoproteins are their ability to reversibly bind to lipoprotein surfaces, their content and type of amphipathic ␣-helices (6), and their ability to form discoidal lipoprotein particles (8).The association of apolipoproteins with lipids is a complex process that involves several steps including protein conformational changes and the disruption of the structure of the phospholipid monolayer or bilayer. Because of this complexity, the elucidation of the limiting steps and energy barriers involved in the formation of lipoproteins is also a complex task. Two processes were clearly distinguished in binding of apoLp-III to a planar lipid bilayer (9); the first process consisted in an adsorption of apoLp-III to the phospholipid bilayer, whereas the second process involved the insertion of the protein into the lipid bilayer and the formation of lipoprotein complexes (9). A recent study with disulfide mutants of apoE has shown that three...