Structure and function of human phospholamban pentamer

人受磷蛋白五聚体的结构和功能

• 批准号: 7083077
• 负责人: JAMES Jeiwen CHOU
• 金额: $ 33.9万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-15 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7083077
• 关键词: calcium transporting ATPase; cardiac myocytes; conformation; detergents; fluorescence; gene expression; ion transport; lipid bilayer membrane; membrane channels; membrane permeability; micelles; muscle contraction; muscle relaxation; nuclear magnetic resonance spectroscopy; phospholamban; phosphorylation; protein protein interaction; protein structure function; sarcoplasmic reticulum

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.

描述（由申请人提供）：心脏病是美国第一大杀手。心脏的逐搏功能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。人受磷蛋白 (PLB) 在​​心肌细胞的 SR 膜中以 30 kDa 同五聚体的形式表达，通过依赖于其磷酸化的机制调节细胞钙水平。我们提案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NMR测定洗涤剂胶束中去磷酸化PLB五聚体的结构，并验证小双胶束脂质双层环境中的结构。 (2) 描述 PLB 五聚体在磷酸化后通过 NMR 偶极耦合产生的方向限制而发生的结构变化。这涉及开发顺磁对准系统，用于精确测量在 bicelles 中重组的 PLB 五聚体的偶极耦合。 (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+ATPase) 之间的抑制相互作用。 (4) 使用脂质体测定和其他电生理学技术进行通道电导测量，研究 PLB 五聚体除了作为 SR 钙泵调节器的既定作用之外，是否还充当离子通道。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多少，以及它们在磷酸化时如何变化。心肌细胞中钙循环不足会导致心脏功能严重恶化。对PLB结构和机制的透彻理解将为新型心脏治疗提供新的机遇，因为这将使我们能够合理设计通过PLB的作用微调钙循环的方法。