Examination of the structure/function relationship in the amphipathic biopolymer, apolipophorin-III
Exchangeable apolipoproteins are proteins that reversibly bind lipoprotein particles to facilitate their transport in vivo. The structure-function relationship of apolipophorin-III (apo-III), the only known insect exchangeable apolipoprotein, has been investigated by examining the association of this protein with amphiphilic materials in vitro. Recombinant DNA technology has been used to synthesize and specifically modify the protein in E. coli. The importance of a conserved leucine residue, reported in previous literature to be essential for apo-III to bind lipids, has been evaluated through site-directed mutagenesis. A unique cysteine was substituted in place of the conserved leucine at position 31 in recombinant apo-III (L31C protein). Incorporation of this amino acid provides a site for specific modification, for example, fluorescent labeling. In addition, the leucine-to-cysteine mutation allows the covalent dimerization of the apo-III mutant via a disulfide bond. The solution behavior of the wild-type apo-III and the L31C proteins has been examined. The cysteine mutation has resulted in slight differences in the surface hydrophobicity and the isoelectric point of the L31C protein from the wild type apo-III. Wild type apo-III, L31C monomer, L31C dimer, and fluorescently labeled L31C associate with dimyristoylphosphatidylcholine (DMPC) vesicles destabilizing them and reducing the turbidity of the suspension. The structures of the phospholipid: protein complexes formed by the wild type apo-III, L31C monomer and dimer resemble the disc-like structures formed by other apolipoproteins in the presence of DMPC. Because the mutation of the conserved leucine seems to affect the solution behavior and surface hydrophobicity of apo-III, this residue is likely to be solvent-exposed. However, the similar behaviors of the wild type protein, the L31C monomer and dimer with respect to the binding of phospholipid vesicles suggest that this residue is not solely responsible for the ability of the protein to bind to hydrophobic or amphiphilic interfaces.