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。我们的研究将集中在视网膜恢复蛋白，参与癌症相关的视网膜病变;鸟苷酸环化酶激活蛋白（GCAP）;与常染色体显性视锥细胞营养不良; centrins，控制光感受器中的蛋白质易位;和钙结合蛋白-4（CaBP 4），涉及先天性静止性夜盲症。通过继续深入研究视网膜恢复蛋白和GCAP蛋白，并将其范围扩大到神经元同源物和蛋白质靶点，我们希望获得对钙传感器蛋白在信号转导和疾病过程中如何运作的原子级理解。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3个：（1）确定与视网膜鸟苷酸环化酶（RetGCs）结合的GCAP蛋白的原子水平结构，以阐明RetGCs的Ca 2+依赖性激活机制，从而为理解视力恢复和视网膜退行性疾病的机制提供结构基础;（2）确定与T？为了解光依赖性蛋白质转位机制及其在光适应中的作用提供结构基础;（3）确定视网膜钙传感蛋白（CaBP 4）的原子水平结构及其与视杆突触电压门控Ca 2+通道（CaV 1. 4）的结构相互作用，以了解与视网膜疾病相关的离子通道的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 focus on retinal recoverin, involved in cancer-associated retinopathy; guanylate cyclase activating proteins (GCAPs); linked to autosomal dominant cone dystrophy; centrins, that control protein translocation in photoreceptors; and calcium binding protein-4 (CaBP4), implicated in congenital stationary night blindness. By continuing our intensive study of retinal recoverin and the GCAP 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 centrin-1 bound to T¿? to provide a structural basis for understanding the mechanism of light-dependent protein translocation and its role in light adaptation; (3) Determine atomic-level structures of the retinal calcium sensor protein (CaBP4) and its structural interaction with voltage-gated Ca2+ channels (CaV1.4) at the rod synapse to understand the Ca2+-dependent regulatory mechanism of ion channels linked to retinal diseases.