ONLY THE 2 END HELIXES OF 8 TANDEM AMPHIPATHIC HELICAL DOMAINS OF HUMAN APO A-I HAVE SIGNIFICANT LIPID AFFINITY - IMPLICATIONS FOR HDL ASSEMBLY

Human apolipoprotein A-I (ape A-I) possesses multiple tandem repeating 22-mer amphipathic alpha-helixes. Computer analysis and studies of mo del synthetic peptides and recombinant protein-lipid complexes of phos pholipids have suggested that apo A-I interacts with HDL surface lipid s through cooperation among its individual amphipathic helical domains . To delineate the overall lipid-associating properties of apo A-I, th e first step is to understand the lipid-associating properties of indi vidual amphipathic helical domains. To this end, we synthesized and st udied each of the eight tandem repeating 22-mer domains of apo A-I: re sidues 44-65, 66-87, 99-120, 121-142, 143-164, 165-186, 187-208, and 2 20-241. Among the 22-mers, only the N- and C-terminal peptides (44-65 and 220-241) were effective in clarifying multilamellar vesicles (MLVs ) of dimyristoylphosphatidylcholine (DMPC). These two peptides also ex hibited the highest partition coefficient into almitoyl-2-oleoyl-sn-gl ycero-3-phosphatidylcholine liposomes, the highest exclusion pressure for penetration into an egg yolk phosphatidylcholine monolayer, and th e greatest reduction in the enthalpy of the gel-to-liquid crystalline phase transition of DMPC MLVs. These results suggest that the strong, lipid-associating properties of apo A-I are localized to the N- and C- terminal amphipathic domains. Although each of the eight peptides stud ied has an amphipathic structure, models based on changes in residual effective amino acid hydrophobicity resulting from differing depths of helix penetration into the lipid are best able to explain the high li pid affinity possessed by the two terminal domains. Differential scann ing calorimetry (DSC) studies showed that on a molar basis, apo A-I is about 10 times more effective than the most effective peptide analyze d in reducing the enthalpy of the gel-to-liquid crystalline phase tran sition of DMPC MLVs. Because previous proteolysis experiments coupled with the present DSC results suggest that the lipid-associating domain s of apo A-I are distributed throughout the length of the 243 amino ac id residues, we propose that the terminal amphipathic helical domains are involved in the initial binding of apo A-I to the lipid surface to form HDL particles, followed by cooperative binding of the middle six amphipathic helical domains, perhaps aided by salt-bridge formation b etween adjacent helixes arranged in an antiparallel orientation.