Depolymerization of phospholamban in the presence of calcium pump: a fluorescence energy transfer study.
Depolymerization of phospholamban in the presence of calcium pump: a fluorescence energy transfer study.
复制标题
钙泵存在下受磷蛋白的解聚：荧光能量转移研究。
DOI：
10.1021/bi981795d
复制
发表时间：
1999
期刊：
影响因子：
2.9
通讯作者：
Thomas,DD
中科院分区：
文献类型：
--
作者：
Reddy,LG;Jones,LR;Thomas,DD
Phospholamban (PLB) 是一种 52 个氨基酸的蛋白质，通过 β-肾上腺素能刺激介导的 PLB 磷酸化调节心脏肌浆网 (SR) 中的 Ca-ATP 酶（钙泵）。 PLB 在 SDS-PAGE 上的迁移率表明它是同源五聚体，并且有人提出 PLB 的五聚体结构对其调节功能很重要。然而，PLB 的寡聚结构必须在其天然环境（含有 Ca-ATP 酶的脂质双层）中确定。在这里，我们使用荧光能量转移（FET）来研究在不存在和存在 Ca-ATPase 的情况下重建的 SDS 和二油酰磷脂酰胆碱（DOPC）脂质双层中 PLB 的寡聚结构。 PLB 被标记，特别是在细胞质结构域的 Lys 3 处，用胺反应性荧光供体/受体对进行标记。 SDS 溶液和 DOPC 脂质双层中 PLB 供体标记亚基和受体标记亚基之间的 FET 表明存在 PLB 寡聚物。 FET 效率对 DOPC 双层中受体标记 PLB 比例的依赖性表明，它主要是具有 9−11 个亚基的低聚物，其中约 10% 的 PLB 作为单体，相邻 PLB 亚基上的染料之间的距离为 0.9 ± 0.1 nm。当用纯化的 Ca-ATP 酶重构标记的 PLB 时，FET 表明 PLB 解聚成平均有 5 个亚基的较小寡聚物，同时单体比例增加至 30−40%，亚基间距离加倍。我们得出结论，PLB 主要作为膜中的寡聚物存在，并且 Ca-ATP 酶影响该寡聚物的结构，但 Ca-ATP 酶优先与单体和/或小寡聚物结合。这些结果表明PLB的活性抑制物质是具有少于5个亚基的单体或寡聚物。
Phospholamban (PLB), a 52-amino acid protein, regulates the Ca-ATPase (calcium pump) in cardiac sarcoplasmic reticulum (SR) through PLB phosphorylation mediated by β-adrenergic stimulation. The mobility of PLB on SDS−PAGE indicates a homopentamer, and it has been proposed that the pentameric structure of PLB is important for its regulatory function. However, the oligomeric structure of PLB must be determined in its native milieu, a lipid bilayer containing the Ca-ATPase. Here we have used fluorescence energy transfer (FET) to study the oligomeric structure of PLB in SDS and dioleoylphosphatidylcholine (DOPC) lipid bilayers reconstituted in the absence and presence of Ca-ATPase. PLB was labeled, specifically at Lys 3 in the cytoplasmic domain, with amine-reactive fluorescent donor/acceptor pairs. FET between donor- and acceptor-labeled subunits of PLB in SDS solution and DOPC lipid bilayers indicated the presence of PLB oligomers. The dependence of FET efficiency on the fraction of acceptor-labeled PLB in DOPC bilayers indicated that it is predominantly an oligomer having 9−11 subunits, with ∼10% of the PLB as monomer, and the distance between dyes on adjacent PLB subunits is 0.9 ± 0.1 nm. When labeled PLB was reconstituted with purified Ca-ATPase, FET indicated the depolymerization of PLB into smaller oligomers having an average of 5 subunits, with a concomitant increase in the fraction of monomer to 30−40% and a doubling of the intersubunit distance. We conclude that PLB exists primarily as an oligomer in membranes, and the Ca-ATPase affects the structure of this oligomer, but the Ca-ATPase binds preferentially to the monomer and/or small oligomers. These results suggest that the active inhibitory species of PLB is a monomer or an oligomer having fewer than 5 subunits.