Robia Seth L, Flohr Nicole C, Thomas David D
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Biochemistry. 2005 Mar 22;44(11):4302-11. doi: 10.1021/bi0478446.
We measured in-gel fluorescence anisotropy of phospholamban (PLB) labeled with the biarsenical fluorophore FlAsH 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 A, 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.
我们在心肌肌浆网钙泵受磷蛋白（PLB）胞质结构域的三个不同位点上，测量了用双砷荧光团FlAsH标记的PLB的凝胶内荧光各向异性。FlAsH-四半胱氨酸PLB的6 kDa单体条带显示出高各向异性（r = 0.29），反映出在FlAsH荧光寿命的纳秒时间尺度上无同源转移且迁移率低（S = 0.85）。同一条泳道内的30 kDa条带（五聚体PLB）显示出低各向异性，表明PLB亚基之间存在五聚体内荧光能量同源转移。位于-6、5或23位残基处的FlAsH标记显示出荧光去极化的梯度模式，分别对应于46±2、38±4和<25 Å的共振能量转移半径。随着加热或荧光光漂白，五聚体各向异性增加，趋向于与单体PLB测定的最大值相似的值。在体外和体内，在用FlAsH和双砷荧光团ReAsH共标记的PLB五聚体中也观察到了荧光共振能量异源转移。体外异源转移效率按标记位置呈梯度变化，与同源转移结果一致。计算得到的转移半径与根据NMR溶液结构构建的心肌肌浆网钙泵受磷蛋白五聚体计算机分子模型预测的距离相当。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中央束急剧向外弯曲。
