Hauser Paul S, Raussens Vincent, Yamamoto Taichi, Abdullahi Gezman E, Weers Paul M M, Sykes Brian D, Ryan Robert O
Center for Prevention of Obesity, Diabetes, and Cardiovascular Disease, Children's Hospital Oakland Research Institute, Oakland, CA 94609, USA.
J Lipid Res. 2009 Aug;50(8):1548-55. doi: 10.1194/jlr.M800554-JLR200. Epub 2008 Dec 19.
Apolipoprotein E (apoE) is an exchangeable apolipoprotein that functions as a ligand for members of the LDL receptor family, promoting lipoprotein clearance from the circulation. Productive receptor binding requires that apoE adopt an LDL receptor-active conformation through lipid association, and studies have shown that the 22 kDa N-terminal (NT) domain (residues 1-183) of apoE is both necessary and sufficient for receptor interaction. Using intein-mediated expressed protein ligation (EPL), a semisynthetic apoE3 NT has been generated for use in structure-function studies designed to probe the nature of the lipid-associated conformation of the protein. Circular dichroism spectroscopy of EPL-generated apoE3 NT revealed a secondary structure content similar to wild-type apoE3 NT. Likewise, lipid and LDL receptor binding studies revealed that EPL-generated apoE3 NT is functional. Subsequently, EPL was used to construct an apoE3 NT enriched with 15N solely and specifically in residues 112-183. 1H-15N heteronuclear single quantum correlation spectroscopy experiments revealed that the ligation product is correctly folded in solution, adopting a conformation similar to wild-type apoE3-NT. The results indicate that segmental isotope labeling can be used to define the lipid bound conformation of the receptor binding element of apoE as well as molecular details of its interaction with the LDL receptor.
载脂蛋白E（apoE）是一种可交换的载脂蛋白，作为低密度脂蛋白受体家族成员的配体发挥作用，促进脂蛋白从循环中清除。有效的受体结合要求apoE通过脂质结合采用低密度脂蛋白受体活性构象，并且研究表明，apoE的22 kDa N端（NT）结构域（第1 - 183位氨基酸残基）对于受体相互作用既必要又充分。利用内含肽介导的表达蛋白连接（EPL）技术，已生成一种半合成的apoE3 NT，用于结构 -功能研究，旨在探究该蛋白脂质相关构象的本质。对EPL生成的apoE3 NT进行圆二色光谱分析，结果显示其二级结构含量与野生型apoE3 NT相似。同样，脂质和低密度脂蛋白受体结合研究表明，EPL生成的apoE3 NT具有功能。随后，利用EPL构建了仅在第112 - 183位氨基酸残基特异性富集15N的apoE3 NT。1H - 15N异核单量子相关光谱实验表明，连接产物在溶液中正确折叠，采用与野生型apoE3 - NT相似的构象。结果表明，片段同位素标记可用于确定apoE受体结合元件的脂质结合构象及其与低密度脂蛋白受体相互作用的分子细节。
