Structure and function of human phospholamban pentamer

描述（由申请人提供）：心脏病是美国的头号杀手。心脏的搏动功能在很大程度上是由心肌细胞的细胞质和肌浆网（SR）之间的钙循环控制的。人受磷蛋白（PLB）在心肌细胞的SR膜中表达为30 kDa的同型五聚体，其中它通过依赖于其磷酸化的机制调节细胞钙水平。我们的目标是在原子水平上了解磷酸化和去磷酸化如何影响PLB五聚体的结构和功能。该提案包括四个具体目标。(1)通过溶液核磁共振测定了洗涤剂胶束中脱磷酸化PLB五聚体的结构，并验证了其在小胶束脂双层环境中的结构。(2)描绘PLB五聚体在通过NMR偶极偶联衍生的取向限制磷酸化后的结构变化。这涉及到一个顺磁对准系统的发展，用于精确测量的偶极耦合的PLB五聚体在bicelles重组。(3)通过NMR化学位移扰动、荧光能量转移和潜在的晶体学方法表征PLB五聚体和SR钙泵（Ca2+ ATP酶）之间的抑制性相互作用。(4)使用脂质体测定和其他用于通道电导测量的电生理学技术，研究PLB五聚体除了作为SR钙泵的调节剂的既定作用外，是否还作为离子通道发挥功能。这些实验旨在回答以下问题：PLB通道的离子渗透性和选择性是什么，以及它们在磷酸化后如何变化。心肌细胞中钙循环不足导致心脏功能严重恶化。对PLB结构和机制的深入了解将为新型心脏治疗提供新的机会，因为它将使我们能够合理地设计通过PLB的作用微调钙循环的方法。

DESCRIPTION (provided by applicant): Heart disease is the number 1 killer in the United States. The beat-to-beat function of heart is largely controlled by the cycling of calcium between the cytoplasm and sarcoplasmic reticulum (SR) of cardiomyocytes, the cells of heart muscle. Human phospholamban (PLB) is expressed in the SR membrane of cardiomyocytes as a 30 kDa homopentamer, where it regulates cellular calcium level by a mechanism that depends on its phosphorylation. The goal of our proposal is to understand at an atomic level how phosphorylation and de-phosphorylation affect the structure and function of the PLB pentamer. The proposal consists of four specific aims. (1) Determine by solution NMR the structure of dephosphorylated PLB pentamer in detergent micelles, and validate the structure in the lipid bilayer environment of small bicelles. (2) Delineate the structural changes in the PLB pentamer upon phosphorylation by orientation restraints derived from NMR dipolar couplings. This involves the development of a paramagnetic alignment system for accurate measurements of dipolar couplings for the PLB pentamer reconstituted in bicelles. (3) Characterize the inhibitory interaction between the PLB pentamer and the SR calcium pump (Ca2+ATPase) by NMR chemical shift perturbation, fluorescence energy transfer, and potentially crystallographic methods. (4) Investigate, using liposome assays and other electrophysiology techniques for channel conductance measurement, whether the PLB pentamer functions as an ion channel, in addition to its established role as a regulator of the SR calcium pumps. The experiments are designed to answer the following questions: what is the ion permeability and selectivity of the PLB channel and how are they changed upon phosphorylation. Inadequate calcium cycling in cardiomyocytes leads to severe deterioration of heart function. A thorough understanding of PLB structure and mechanism will offer new opportunities for novel cardiac therapy, as it will allow us to rationally design methods for fine-tuning calcium cycling through the actions of PLB.