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（心肌细胞）的细胞质和肌浆网（SR）之间的钙循环控制的。人磷蛋白（PLB）在心肌细胞SR膜中作为30 kDa的同型五聚体表达，通过依赖于其磷酸化的机制调节细胞钙水平。我们建议的目标是在原子水平上理解磷酸化和去磷酸化如何影响PLB五聚体的结构和功能。该建议包括四个具体目标。(1)通过溶液核磁共振测定去磷酸化PLB五聚体在洗涤剂胶束中的结构，并验证其在小单束的脂质双分子层环境中的结构。(2)通过核磁共振偶极偶联的取向约束来描述PLB五聚体在磷酸化后的结构变化。这涉及到一个顺磁对准系统的发展，用于精确测量偶极耦合的PLB五聚体重构在自行车。(3)通过核磁共振化学位移摄动、荧光能量转移和潜在的晶体学方法表征PLB五聚体与SR钙泵（Ca2+ atp酶）之间的抑制相互作用。(4)利用脂质体分析和其他电生理学技术来测量通道电导，研究PLB五聚体除了作为SR钙泵的调节剂之外，是否还具有离子通道的功能。该实验旨在回答以下问题：什么是PLB通道的离子通透性和选择性，以及它们在磷酸化后是如何变化的。心肌细胞钙循环不足导致心功能严重恶化。深入了解PLB的结构和机制将为新的心脏治疗提供新的机会，因为它将使我们能够合理设计通过PLB的作用来微调钙循环的方法。

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.