Pyle L E, Sawyer W H, Fujiwara Y, Mitchell A, Fidge N H
Baker Medical Research Institute, Prahran, Australia.
Biochemistry. 1996 Sep 17;35(37):12046-52. doi: 10.1021/bi9609073.
Utilizing the Escherichia coli/pGex vector expression system incorporating a thrombin cleavage site, full-length (residues -6-243) and truncated forms of proapolipoprotein AI (proapoAI), terminating at amino acid residues 222, 210, 150, and 135, were purified to levels of at least 5 mg/L, after thrombin cleavage. Assessed by circular dichroism, the helical contents of L-alpha-dimyristoylphosphatidylcholine-associated forms of human plasma-derived apolipoprotein AI (apoAI) and recombinant proapoAI were comparable, being 69% and 65%, respectively. Circular dichroism measurements of the lipid-associated complexes of the truncated forms showed that between the sequence of residues 150-222 no additional 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 region. While tryptophan residues were more than 86% accessible, as assessed by iodide quenching, in the two truncated forms, proapoAI-6-135 and proapoAI-6-150, for both free and complexed protein, this figure fell to about 50% for full-length recombinant proapoAI, further indicating the influence of the carboxyl terminus on the structure of the whole protein. While cross-linking human plasma apoAI in solution with dithiobis-(succinimidyl propionate) revealed high molecular weight oligomers by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, recombinant proapoAI did not strongly form complexes larger than trimers. None of the truncated proapoAI molecules formed oligomers larger than trimers. The shortest form, proapoAI-6-135, only dimerized. Initial results from lecithin:cholesterol acyltransferase activation (apoAI peptide concentration 0.2 microM) indicated that truncation of the 21 carboxy-terminal amino acids resulted in a drop of approximately 53% in activation and 33 residues a drop of 67% relative to the full-length protein. Overall these results indicate the important influence of the carboxyl terminus on the structure of apoAI.
利用包含凝血酶切割位点的大肠杆菌/pGex载体表达系统，全长（残基-6至243）和截短形式的载脂蛋白AI前体（proapoAI），分别在氨基酸残基222、210、150和135处终止，在凝血酶切割后，纯化至至少5mg/L的水平。通过圆二色性评估，人血浆来源的载脂蛋白AI（apoAI）和重组proapoAI与L-α-二肉豆蔻酰磷脂酰胆碱相关形式的螺旋含量相当，分别为69%和65%。截短形式的脂质相关复合物的圆二色性测量表明，在残基150-222序列之间，直到分子中存在羧基末端序列才获得额外的螺旋性，这表明蛋白质的羧基末端是该中心区域内螺旋形成所必需的。通过碘化物猝灭评估，在两种截短形式proapoAI-6-135和proapoAI-6-150中，无论是游离蛋白还是复合蛋白，色氨酸残基的可及性均超过86%，而全长重组proapoAI的这一数字降至约50%，进一步表明羧基末端对整个蛋白质结构的影响。虽然用二硫代双（琥珀酰亚胺丙酸酯）在溶液中交联人血浆apoAI通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示出高分子量寡聚体，但重组proapoAI并没有强烈形成大于三聚体的复合物。截短的proapoAI分子均未形成大于三聚体的寡聚体。最短的形式proapoAI-6-135仅形成二聚体。卵磷脂：胆固醇酰基转移酶激活的初步结果（apoAI肽浓度0.2μM）表明，截短21个羧基末端氨基酸导致激活下降约53%，相对于全长蛋白截短33个残基导致激活下降67%。总体而言，这些结果表明羧基末端对apoAI结构具有重要影响。
