Center for Prevention of Obesity, Cardiovascular Disease and Diabetes, Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, California 94609, USA.
Biochemistry. 2010 Jun 15;49(23):4821-6. doi: 10.1021/bi1005859.
Apolipoprotein (apo) A-V is a 343-residue, multidomain protein that plays an important role in regulation of plasma triglyceride homeostasis. Primary sequence analysis revealed a unique tetraproline sequence (Pro293-Pro296) near the carboxyl terminus of the protein. A peptide corresponding to the 48-residue segment beyond the tetraproline motif was generated from a recombinant apoA-V precursor wherein Pro295 was replaced by Met. Cyanogen bromide cleavage of the precursor protein, followed by negative affinity chromatography, yielded a purified peptide. Nondenaturing polyacrylamide gel electrophoresis verified that apoA-V(296-343) solubilizes phospholipid vesicles, forming a relatively heterogeneous population of reconstituted high-density lipoprotein with Stokes' diameters >17 nm. At the same time, apoA-V(296-343) failed to bind a spherical lipoprotein substrate in vitro. Far-UV circular dichroism spectroscopy revealed the peptide is unstructured in buffer yet adopts significant alpha-helical secondary structure in the presence of the lipid mimetic solvent trifluoroethanol (TFE; 50% v/v). Heteronuclear multidemensional NMR spectroscopy experiments were conducted with uniformly (15)N- and (15)N/(13)C-labeled peptide in 50% TFE. Peptide backbone assignment and secondary structure prediction using TALOS+ reveal the peptide adopts alpha-helix secondary structure from residues 309 to 334. In TFE, apoA-V(296-343) adopts an extended amphipathic alpha-helix, consistent with a role in lipoprotein binding as a component of full-length apoA-V.
载脂蛋白（apo）A-V 是一种 343 个残基的多功能蛋白，在调节血浆甘油三酯稳态方面发挥着重要作用。 一级序列分析显示，该蛋白羧基末端附近存在一个独特的四脯氨酸序列（Pro293-Pro296）。 从含有 Pro295 被 Met 取代的重组 apoA-V 前体中生成了一个对应于四脯氨酸基序之后 48 个残基片段的肽。 前体蛋白的氰溴酸裂解，然后进行负亲和层析，得到了纯化的肽。 非变性聚丙烯酰胺凝胶电泳证实 apoA-V（296-343）可溶解磷脂囊泡，形成相对异质的再构成高密度脂蛋白群体，其斯托克斯直径>17nm。 同时，apoA-V（296-343）未能在体外结合球形脂蛋白底物。 远紫外圆二色光谱表明该肽在缓冲液中无结构，但在脂质模拟溶剂三氟乙醇（TFE；50%v/v）中采用显著的α-螺旋二级结构。 用均匀标记的（15）N-和（15）N/（13）C-标记肽进行异核多维 NMR 光谱实验在 50%TFE 中进行。 使用 TALOS+进行肽骨架分配和二级结构预测表明，肽从残基 309 到 334 采用α-螺旋二级结构。 在 TFE 中，apoA-V（296-343）采用扩展的两亲性α-螺旋，这与作为全长 apoA-V 组成部分的脂蛋白结合作用一致。
