THE ROLE OF APOLIPOPROTEIN AI DOMAINS IN LIPID-BINDING

Apolipoprotein AI (apoAI) is the principal protein constituent of high density lipoproteins and it plays a key role in human cholesterol hom eostasis; however, the structure of apoAI is not clearly understood. T o test the hypothesis that apoAI is organized into domains, three dele tion mutants of human apoAI expressed in Escherichia coli were studied in solution and in reconstituted high density lipoprotein particles. Each mutant lacked one of three specific regions that together encompa ss almost the entire 243 aa sequence of native apoAI (apoAI Delta 44-1 26, apoAI Delta 139-170, and apoAI Delta 190-243), Circular dichroism spectroscopy showed that the alpha-helical content of lipid-free apoAI Delta 44-126 was 27% while the other mutants and native apoAI average d 55 +/- 2%, suggesting that the missing N-terminal portion contains m ost of the alpha-helical structure of lipid-free apoAI. ApoAI Delta 44 -126 exhibited the largest increase in alpha-helix upon lipid binding (125% increase versus an average of 25% for the others), confirming th e importance of the C-terminal half of apoAI in lipid binding, Denatur ation studies showed that the N-terminal half of apoAI is primarily re sponsible for alpha-helix stability in the lipid-free state, whereas t he C terminus is required for alpha-helix stability when lipid-bound, We conclude that the N-terminal half (aa 44-126) of apoAI is responsib le for most of the alpha-helical structure and the marginal stability of lipid-free apoAI while the C terminus (aa 139-243) is less organize d, The increase in alpha-helical content observed when native apoAI bi nds lipid results from the formation of alpha-helix primarily in the C -terminal half of the molecule.