Membrane targeting calcium sensors in vision

 描述（由申请人提供）：总体目标是开发核磁共振（NMR）技术，并将其与其他实验方法结合使用，以阐明参与视觉和其他信号转导过程中光转导的选定膜靶向蛋白的分子结构和生理功能。在接下来的五年中，我们将使用核磁共振（NMR），荧光，微量热法，自旋标记EPR，X射线晶体学和计算分析来描述神经元钙传感器蛋白（钙肉豆蔻酰开关）家族的结构，动力学和机制，这些蛋白在钙信号传导中作为膜靶向调节剂，并与视网膜和神经系统疾病有关。我们的研究将确定以下结构基础：（1）钙依赖性激活视网膜鸟苷酸环化酶（RetGC）的GCAP 1，遗传上与常染色体显性视锥细胞营养不良有关;（2）钙诱导的抑制光感受器环核苷酸门控（CNG）通道的CNG-调制蛋白;（3）钙结合蛋白4（CaBP 4）对视网膜L型钙通道（CaV1.4）的钙依赖性激活，与先天性静止性夜盲症有关。通过继续深入研究视网膜钙传感器蛋白，并扩大其范围，包括神经元同源物和蛋白质靶点，我们希望获得一个原子水平的钙传感器蛋白如何在信号转导和疾病过程中运作的理解。特别是，我们想了解共价连接的肉豆蔻酰基如何与钙结合位点和靶蛋白协同工作，以指导这一家族的蛋白质特异性膜结合靶点。具体目标有三：（1）确定视网膜鸟苷酸环化酶（RetGCs）结合的GCAP蛋白的原子水平结构，阐明RetGCs的Ca ~（2+）依赖性激活机制，为理解视力恢复和视网膜变性疾病的机制提供结构基础;（2）确定与CNG通道结合的CNG-调制蛋白的结构，为理解视锥细胞光适应机制提供结构基础;（3）确定视杆突触处与视网膜L型电压门控Ca 2+通道（CaV1.4）结合的视网膜钙传感器蛋白（CaBP 4）的原子水平结构，以了解与先天性静止性夜盲相关的离子通道的Ca 2+依赖性调节机制。

 DESCRIPTION (provided by applicant): The overall objectives are to develop nuclear magnetic resonance (NMR) techniques and use them in concert with other experimental approaches to elucidate the molecular structure and physiologic functions of selected membrane-targeting proteins involved in phototransduction in vision and other signal transduction processes. During the next five years, we will use nuclear magnetic resonance (NMR), fluorescence, microcalorimetry, spin-label EPR, x-ray crystallography, and computational analysis to delineate the structure, dynamics and mechanisms of a family of neuronal calcium sensor proteins (calcium-myristoyl switches) that serve as membrane- targeting regulators in calcium signaling and are linked to retinal and neurological diseases. Our studies will determine the structural basis of: (1) Ca2+-dependent activation of retinal guanylate cyclase (RetGC) by GCAP1, genetically linked to autosomal dominant cone dystrophy; (2) Ca2+-induced inhibition of photoreceptor cyclic nucleotide gated (CNG) channels by CNG-modulin; and (3) Ca2+- dependent activation of retinal L-type Ca2+ channels (CaV1.4) by calcium binding protein-4 (CaBP4), implicated in congenital stationary night blindness. By continuing our intensive study of retinal calcium sensor proteins and by broadening its scope to encompass neuronal homologs and protein targets, we hope to gain an atomic-level understanding of how calcium sensor proteins operate in signal transduction and disease processes. In particular, we want to understand how covalently attached myristoyl groups work in concert with calcium-binding sites and target proteins to guide this family of proteins to specific membrane-bound targets. The specific aims are 3-fold: (1) Determine atomic-level structures of the GCAP proteins bound to retinal guanylate cyclases (RetGCs) to elucidate the Ca2+-dependent activation mechanism of RetGCs and thus provide a structural basis for understanding mechanisms of visual recovery and retinal degenerative diseases; (2) Determine structures of CNG-modulin bound to CNG channels to provide a structural basis for understanding the mechanism of light-adaptation in cone photoreceptors; (3) Determine atomic-level structures of the retinal calcium sensor protein (CaBP4) bound to the retinal L-type voltage-gated Ca2+ channel (CaV1.4) at the rod synapse to understand the Ca2+-dependent regulatory mechanism of ion channels linked to congenital stationary night blindness.