STRUCTURAL AND FUNCTIONAL-PROPERTIES OF FULL-LENGTH AND TRUNCATED HUMAN PROAPOLIPOPROTEIN AI EXPRESSED IN ESCHERICHIA-COLI

Utilizing the Escherichia coli/pGex vector expression system incorpora ting a thrombin cleavage site, full-length (residues -6-243) and trunc ated forms of proapolipoprotein AI (proapoAI), terminating at amino ac id residues 222, 210, 150, and 135, were purified to levels of at leas t 5 mg/L, after thrombin cleat-age, Assessed by circular dichroism, th e helical contents of L-alpha-dimyristoylphosphatidylcholine-associate d forms of human plasma-derived apolipoprotein AI (apoAI) and recombin ant proapoAI were comparable, being 69% and 65%, respectively. Circula r dichroism measurements of tile lipid-associated complexes of the tru ncated forms showed that between the sequence of residues 150-222 no a dditional helicity was gained until the carboxyl-terminal sequence was present in the molecule, indicating that the carboxyl terminus of the protein is required for the formation of helix within this central re gion. While tryptophan residues were more than 86% accessible, as asse ssed by iodide quenching, in the two truncated forms, proapaAI(-6-135) and proapoAI(-6-150), for both free and complexed protein, this figur e fell to about 50% or full-length recombinant proapoAI, further indic ating the influence of the carboxyl terminus on the structure of tile whole protein. While cross-linking human plasma apoAI in solution with dithiobis-(succinimidyl propionate) revealed high molecular weight ol igomers by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, recombinant proapoAI did not strongly form complexes larger than trime rs. None of the truncated proapoAI molecules formed oligomers larger t han trimers. The shortest form, proapoAI(-6-135), only dimerized. Init ial results from lecithin:cholesterol acyltransferase activation (apoA I peptide concentration 0.2 mu M) indicated that truncation of the 21 carboxy-terminal amino acids resulted in a drop of approximately 53% i n activation and 33 residues a drop of 67% relative to the full-length protein. Overall these results indicate the important influence of th e carboxyl terminus on the structure of apoAI.