Department of Cell and Molecular Physiology, Loyola University Chicago, 2160 South First Avenue, Maywood, IL60153, USA.
J Mol Biol. 2010 Sep 10;402(1):210-6. doi: 10.1016/j.jmb.2010.07.023. Epub 2010 Jul 17.
To investigate the regulation of SERCA1a [sarco(endo)plasmic reticulum calcium ATPase] and SERCA2a calcium pump isoforms by phospholamban (PLB), we quantified PLB-SERCA interactions by fluorescence resonance energy transfer (FRET) in live cells. For both SERCA1a and SERCA2a, FRET to PLB increased with increasing protein expression level to a maximum value corresponding to a probe separation distance of 64 A. The data indicate that the respective regulatory complexes assume the same overall quaternary conformation. However, FRET measurements also revealed that PLB has a 50% higher apparent affinity for SERCA1a relative to SERCA2a. The results suggest that despite the structural similarities of the respective regulatory complexes, there is preferential binding of PLB to SERCA1a over SERCA2a. This apparent selectivity may have implications for biochemical studies in which SERCA1a is used as a substitute for SERCA2a. It may also be an important strategic consideration for therapeutic overexpression of SERCA isoforms in cardiac muscle.
为了研究肌浆网钙 ATP 酶（SERCA）1a 和 SERCA2a 钙泵同工型受磷蛋白（PLB）的调节，我们通过荧光共振能量转移（FRET）在活细胞中定量了 PLB-SERCA 相互作用。对于 SERCA1a 和 SERCA2a，FRET 到 PLB 的增加与蛋白表达水平的增加成正比，最大值对应于探针分离距离为 64A。数据表明，各自的调节复合物具有相同的整体四级构象。然而，FRET 测量还表明，PLB 对 SERCA1a 的表观亲和力相对于 SERCA2a 高 50%。结果表明，尽管各自的调节复合物具有结构相似性，但 PLB 优先与 SERCA1a 结合而不是 SERCA2a。这种明显的选择性可能对使用 SERCA1a 作为 SERCA2a 替代物进行生化研究具有影响。对于在心肌中过表达 SERCA 同工型的治疗方法，这也可能是一个重要的策略考虑因素。
