A fluorescence energy transfer method for analyzing protein oligomeric structure: Application to phospholamban
A fluorescence energy transfer method for analyzing protein oligomeric structure: Application to phospholamban
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DOI：
10.1016/s0006-3495(99)77411-4
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发表时间：
1999-05-01
影响因子：
3.4
通讯作者：
Thomas, DD
中科院分区：
文献类型：
--
作者：
Li, M;Reddy, LG;Thomas, DD
我们开发了一种使用荧光能量转移 (FET) 来分析蛋白质寡聚结构的方法。两个蛋白质群体分别用荧光供体和受体标记，然后以规定的供体/受体比例混合。假设环形同源低聚物中的蛋白质亚基之间随机混合和缔合，使用理论模拟来确定 FET 对亚基数量、不同亚基上标记位点之间的距离以及保持单体的亚基分数的依赖性。通过测量 FET 作为供体/受体比率的函数，可以在其值的很大范围内解析寡聚结构的上述参数。我们使用这种方法研究了受磷蛋白 (PLB) 的寡聚结构，PLB 是心脏肌浆网 (SR) 中的一种 52 个氨基酸的蛋白质。 PLB 的磷酸化调节 SR Ca-ATP 酶。由于PLB在十二烷基硫酸钠聚丙烯酰胺凝胶电泳上主要以同五聚体形式存在，因此有人提出PLB的五聚体结构对其调节功能很重要。然而，必须通过直接确定脂膜中 PLB 的寡聚结构来检验这一假设。为了实现这一目标，PLB 在细胞质结构域的 Lys-3 处被标记，并具有两个不同的胺反应性供体/受体对，这给出了非常相似的 FET 结果。在洗涤剂溶液中，除非先将样品煮沸以促进亚基混合，否则不会观察到 FET。在脂质双层中，在 25 摄氏度且未沸腾的情况下观察到 FET，表明膜中 PLB 亚基之间存在动态平衡。 FET数据的分析表明，染料标记的PLB主要是具有至少8个亚基的寡聚物，7-23%的PLB亚基是单体，并且相邻PLB亚基上的染料之间的距离约为10埃。在 SDS-PAGE 上作为单体运行的 PLB (L37A) 的点突变显示没有能量转移，证实了其在膜中的单体状态。我们得出结论，FET 是分析 PLB 寡聚结构的强大方法，并且该方法适用于其他寡聚蛋白。
We have developed a method using fluorescence energy transfer (FET) to analyze protein oligomeric structure. Two populations of a protein are labeled with fluorescent donor and acceptor, respectively, then mixed at a defined donor/acceptor ratio. A theoretical simulation, assuming random mixing and association among protein subunits in a ring-shaped homo-oligomer, was used to determine the dependence of FET on the number of subunits, the distance between labeled sites on different subunits, and the fraction of subunits remaining monomeric. By measuring FET as a function of the donor/acceptor ratio, the above parameters of the oligomeric structure can be resolved over a substantial range of their values. We used this approach to investigate the oligomeric structure of phospholamban (PLB), a 52-amino acid protein in cardiac sarcoplasmic reticulum (SR). Phosphorylation of PLB regulates the SR Ca-ATPase. Because PLB exists primarily as a homopentamer on sodium dodecyl sulfate polyacrylamide gel electrophoresis, it has been proposed that the pentameric structure of PLB is important for its regulatory function. However, this hypothesis must be tested by determining directly the oligomeric structure of PLB in the lipid membrane. To accomplish this goal, PLB was labeled at Lys-3 in the cytoplasmic domain, with two different amine-reactive donor/acceptor pairs, which gave very similar FET results. In detergent solutions, FET was not observed unless the sample was first boiled to facilitate subunit mixing. In lipid bilayers, FET was observed at 25 degrees C without boiling, indicating a dynamic equilibrium among PLB subunits in the membrane. Analysis of the FET data indicated that the dye-labeled PLB is predominantly in oligomers having at least 8 subunits, that 7-23% of the PLB subunits are monomeric, and that the distance between dyes on adjacent PLB subunits is about 10 Angstrom. A point mutation of PLB (L37A) that runs as monomer on SDS-PAGE showed no energy transfer, confirming its monomeric state in the membrane. We conclude that FET is a powerful approach for analyzing the oligomeric structure of PLB, and this method is applicable to other oligomeric proteins.