Förster transfer recovery reveals that phospholamban exchanges slowly from pentamers but rapidly from the SERCA regulatory complex.
Förster transfer recovery reveals that phospholamban exchanges slowly from pentamers but rapidly from the SERCA regulatory complex.
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Förster 转移恢复表明，受磷蛋白与五聚体的交换缓慢，但与 SERCA 调节复合物的交换迅速。
DOI：
10.1161/circresaha.107.159947
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发表时间：
2007
影响因子：
20.1
通讯作者：
Thomas,DavidD
中科院分区：
文献类型：
--
作者：
Robia,SethL;Campbell,KennethS;Kelly,EileenM;Hou,Zhanjia;Winters,DeborahL;Thomas,DavidD
将受磷蛋白（PLB）或肌浆网Ca ~（2+）-ATP酶（SERCA）与青色荧光蛋白（CFP）融合，并与融合黄色荧光蛋白（YFP）的PLB共表达。使用落射荧光和全内反射荧光显微镜对表达的荧光标记的蛋白质进行成像。通过聚焦激光束选择性地漂白YFP荧光。在YFP光漂白后，靶位点处的CFP荧光增加，表明CFP-SERCA/CFP-PLB和YFP-PLB之间的荧光共振能量转移。由于供体扩散和漂白的YFP-PLB与未漂白的YFP-PLB的交换，增加的供体荧光松弛回到基线，这恢复了荧光共振能量转移。CFP供体的需求，称为Fo Prster转移回收率（FTR），被定量为PLB：SERCA和PLB：PLB膜复合物的PLB亚基交换速率的指数。PLB亚基从PLB：SERCA调节复合物的交换是快速的，显示出扩散限制的FTR（τ=1.4秒）。相反，发现PLB：PLB寡聚复合物在长得多的时间尺度上是稳定的。尽管在膜中存在自由侧向扩散，但它们在80秒内未显示FTR。PLB位置40从异亮氨酸突变为丙氨酸（I40 A-PLB）没有消除PLB：PLB能量转移，但是PLB：PLB复合物的不稳定性从增加的FTR速率（τ=8.4秒）是明显的。通过跨膜半胱氨酸与二酰胺的氧化交联来稳定I40 A-PLB的寡聚体。我们的结论是，PLB交换迅速从其监管复杂的SERCA泵，而从PLB寡聚体复合物的亚基交换是缓慢的，并没有发生在心动周期的时间尺度。
Phospholamban (PLB) or the sarcoplasmic reticulum Ca2+–ATPase (SERCA) were fused to cyan fluorescent protein (CFP) and coexpressed with PLB fused to yellow fluorescent protein (YFP). The expressed fluorescently tagged proteins were imaged using epifluorescence and total internal reflection fluorescence microscopy. YFP fluorescence was selectively bleached by a focused laser beam. CFP fluorescence at the targeted site increased after YFP photobleaching, indicating fluorescence resonance energy transfer between CFP-SERCA/CFP-PLB and YFP-PLB. The increased donor fluorescence relaxed back toward baseline as a result of donor diffusion and exchange of bleached YFP-PLB for unbleached YFP-PLB, which restored fluorescence resonance energy transfer. Requenching of CFP donors, termed Förster transfer recovery (FTR), was quantified as an index of the rate of PLB subunit exchange from the PLB:SERCA and PLB:PLB membrane complexes. PLB subunit exchange from the PLB:SERCA regulatory complex was rapid, showing diffusion-limited FTR (τ=1.4 second). Conversely, PLB:PLB oligomeric complexes were found to be stable on a much longer time scale. Despite free lateral diffusion in the membrane, they showed no FTR over 80 seconds. Mutation of PLB position 40 from isoleucine to alanine (I40A-PLB) did not abolish PLB:PLB energy transfer, but destabilization of the PLB:PLB complex was apparent from an increased FTR rate (τ=8.4 seconds). Oligomers of I40A-PLB were stabilized by oxidative crosslinking of transmembrane cysteines with diamide. We conclude that PLB exchanges rapidly from its regulatory complex with the SERCA pump, whereas subunit exchange from the PLB oligomeric complex is slow and does not occur on the time scale of the cardiac cycle.