Phospholamban pentamer quaternary conformation determined by in-gel fluorescence anisotropy
Phospholamban pentamer quaternary conformation determined by in-gel fluorescence anisotropy
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DOI：
10.1021/bi0478446
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发表时间：
2005-03-22
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：
Robia, SL;Flohr, NC;Thomas, DD
我们测量了凝胶荧光各向异性的磷蛋白（PLB）标记的双砷荧光团HASH在三个不同的网站上的细胞质域。FlAsH-tetracysPLB的6 kDa单体条带显示出高的各向异性（r = 0.29），反映了FlAsH荧光寿命的纳秒时间尺度上的零同质转移和低迁移率（S = 0.85）。相同泳道内的30 kDa条带（五聚体PLB）表现出低各向异性，表明PLB亚基之间的五聚体内荧光能量同源转移。位于残基-6，5或23处的FlAsH标记显示出荧光去极化的渐变图案，分别对应于46 +/-2，38 +/-4和< 25埃的共振能量转移半径。五聚体各向异性增加加热或荧光光漂白朝向最大值类似的单体PLB确定。荧光共振能量异源转移也观察到在体外和体内的PLB五聚体与FlAsH和双砷荧光团ReAsH collabeled。在体外异源转移效率的标记位置，在和谐与homotransfer的结果。计算出的转移半径比较有利的距离预测的计算机分子模型构建的受磷蛋白五聚体从NMR溶液结构。这些数据支持PLB五聚体的螺旋风车模型，其中胞质结构域从螺旋的中心束急剧向外弯曲。
We measured in-gel fluorescence anisotropy of phospholamban (PLB) labeled with the biarsenical fluorophore HASH at three different sites on the cytoplasmic domain. The 6 kDa monomer bands of FlAsH-tetracysPLB showed high anisotropy (r = 0.29), reflecting null homotransfer and low mobility (S = 0.85) on the nanosecond time scale of the FlAsH fluorescence lifetime. 30 kDa bands (pentameric PLB) within the same lanes exhibited low anisotropy, suggesting intrapentameric fluorescence energy homotransfer between PLB subunits. FlAsH labels positioned at residue -6, 5, or 23 showed a graduated pattern of fluorescence depolarization corresponding to resonance energy transfer radii of 46 +/- 2, 38 +/- 4, and < 25 angstrom, respectively. Pentamer anisotropy increased with heating or fluorescence photobleaching toward a maximum value similar to that determined for monomeric PLB. Fluorescence resonance energy heterotransfer was also observed in vitro and in vivo within PLB pentamers colabeled with FlAsH and the biarsenical fluorophore ReAsH. In vitro heterotransfer efficiencies were graduated by labeling position, in harmony with homotransfer results. The calculated transfer radii compare favorably to distances predicted by a computer molecular model of the phospholamban pentamer constructed from NMR solution structures. The data support a helical pinwheel model for the PLB pentamer, in which the cytoplasmic domains bend sharply outward from the central bundle of helices.