Russell Blair A, Horn James V C, Weers Paul M M
Department of Chemistry and Biochemistry, California State University Long Beach, 1250 Bellflower Blvd, Long Beach, CA 90840, United States.
BBA Adv. 2021;1. doi: 10.1016/j.bbadva.2021.100020. Epub 2021 Jul 30.
Apolipophorin III (apoLp-III) from is an exchangeable apolipoprotein with a critical role in lipid transport in insects. The protein is composed of a bundle of five amphipathic α-helices which undergo a large conformational change upon lipid binding. To better understand the apoLp-III lipid binding interaction, the protein was cleaved by cyanogen bromide upon introduction of a S92M mutation, generating an N-terminal fragment corresponding to the first three helices (NT) and a C-terminal fragment of the last two helices (CT). MALDI-TOF analysis of the HPLC purified fragments provided masses of 9863.8 Da for NT and 7497.0 Da for CT demonstrating that the intended fragments were obtained. Circular dichroism spectra revealed a decrease in helical content from 82% for the intact protein to 57% for NT and 41% for CT. The fragments adopted considerably higher α-helical structure in the presence of trifluoroethanol or phospholipids. Equimolar mixing of the two fragments did not result in changes in helical content or tryptophan fluorescence, indicating recombination into the native protein fold did not occur. The rate of protein induced dimyristoylphosphatidylcholine vesicle solubilization increased 15-fold for NT and 100-fold for CT compared to the intact protein. Despite the high activity in phospholipid vesicle interaction, CT did not protect phospholipase-treated low-density lipoprotein from aggregation. In contrast, NT provided protection to lipoprotein aggregation similar to the intact protein, indicating that specific amino acid residues in this part of apoLp-III are essential for lipoprotein binding interaction.
来自[具体来源未提及]的载脂蛋白III（apoLp-III）是一种可交换载脂蛋白，在昆虫脂质转运中起关键作用。该蛋白由一束五个两亲性α螺旋组成，在脂质结合时会发生大的构象变化。为了更好地理解apoLp-III与脂质的结合相互作用，在引入S92M突变后，该蛋白被溴化氰切割，产生了一个对应于前三个螺旋的N端片段（NT）和一个后两个螺旋的C端片段（CT）。对HPLC纯化片段的MALDI-TOF分析显示，NT的质量为9863.8 Da，CT的质量为7497.0 Da，表明获得了预期的片段。圆二色光谱显示，完整蛋白的螺旋含量从82%降至NT的57%和CT的41%。在三氟乙醇或磷脂存在下，这些片段呈现出更高的α螺旋结构。两个片段等摩尔混合并未导致螺旋含量或色氨酸荧光发生变化，这表明未发生重组成天然蛋白折叠的情况。与完整蛋白相比，NT诱导二肉豆蔻酰磷脂酰胆碱囊泡溶解的速率提高了15倍，CT提高了100倍。尽管CT在磷脂囊泡相互作用中具有高活性，但它并不能保护磷脂酶处理的低密度脂蛋白免于聚集。相比之下，NT对脂蛋白聚集的保护作用与完整蛋白相似，这表明apoLp-III这一部分的特定氨基酸残基对于脂蛋白结合相互作用至关重要。
