Abstract

The regulation of the activity and selectivity of phospholipase A2 (PLA2), which is capable of cleaving fatty acid in the second position (sn-2) of the phospholipid, is carried out through the membrane-binding and catalytic sites of the enzyme. For hydrolytic activity, PLA2 must first bind to the phospholipid membrane, and the binding efficiency depends on the composition of the membrane. The membrane-binding site of PLA2 is formed by several tens of amino acids and its composition differs from enzyme to enzyme; hydrophobic and positively charged amino acids play a key role in the interaction. In this work, we investigated the interaction of PLA2 from bee venom with phospholipid bilayers of palmitoyl oleoylphosphatidylcholine (POPC) containing different amounts of palmitoyloleoylphosphatidylglycerol (POPG). On the basis of the measurements of the protein intrinsic fluorescence and the anisotropy of the fluorescence of the lipid probe we propose the construction of lipid–protein interaction maps, which reflect both the efficiency of protein binding and changes in the structure of the membrane. These changes cause alterations in the fluorescence anisotropy of the label, which in turn is a measure of the mobility of the lipid environment of the fluorescent probe. Analysis of interaction maps showed that there is a relationship between lipid mobility and enzyme binding efficiency: the optimum interaction of PLA2 with membranes from a POPC/POPG mixture lies in the region of the highest lipid mobility, and not in the region of the highest negative charge. This dependence complements the existing understanding of the process of recognition of the membrane surface by the enzyme and the selection of lipids by the enzyme already bound to the membrane. The proposed mapping method can be extended to other membrane-active proteins.

中文翻译：

---

使用相互作用图估计 POPC/POPG 膜上的磷脂酶 A2 选择性

摘要

磷脂酶 A2 (PLA2) 的活性和选择性的调节，它能够在第二个位置 ( sn-2)裂解脂肪酸) 的磷脂，是通过酶的膜结合和催化位点进行的。对于水解活性，PLA2 必须首先与磷脂膜结合，结合效率取决于膜的组成。PLA2的膜结合位点由几十个氨基酸组成，其组成因酶而异；疏水性和带正电荷的氨基酸在相互作用中起关键作用。在这项工作中，我们研究了蜂毒中的 PLA2 与含有不同量棕榈酰油酰磷脂酰甘油 (POPG) 的棕榈酰油酰磷脂酰胆碱 (POPC) 磷脂双层的相互作用。基于对蛋白质固有荧光和脂质探针荧光各向异性的测量，我们建议构建脂质-蛋白质相互作用图，这既反映了蛋白质结合的效率，又反映了膜结构的变化。这些变化会导致标记的荧光各向异性发生变化，这反过来又是荧光探针脂质环境迁移率的衡量标准。相互作用图分析表明脂质迁移率和酶结合效率之间存在关系：PLA2 与来自 POPC/POPG 混合物的膜的最佳相互作用位于最高脂质迁移率区域，而不是最高负值区域收费。这种依赖性补充了对酶识别膜表面的过程和已经与膜结合的酶选择脂质的过程的现有理解。所提出的作图方法可以扩展到其他膜活性蛋白。