Membrane targeting calcium sensors in vision

总体目标是开发核磁共振（NMR）技术， 它们与实验功能方法相一致，以阐明分子结构， 参与光转导的选定膜靶向蛋白的生理功能 视觉和其他信号转导过程。在未来五年内，我们将使用核能。 磁共振（NMR）、荧光、微量热法、自旋标记EPR、X射线晶体学， 和计算分析来描绘一个家庭的结构，动力学和机制， 神经元钙传感器蛋白（钙-肉豆蔻酰开关），作为膜- 靶向钙信号传导中的调节剂，并与视网膜和神经系统疾病有关。我们 研究将确定以下的结构基础：（1）视网膜鸟苷酸环化酶（RetGC）的调节， GCAP 1和视网膜变性3（RD 3）蛋白及其在常染色体显性视锥细胞中的作用 营养不良和Leber先天性黑蒙;（2）Ca 2+依赖性感光细胞失活 由钙调蛋白（CaM）控制的环核苷酸门控（CNG）通道;和（3）Ca 2+依赖性 钙结合蛋白4介导的视网膜L型钙通道（CaV1.4）激活 （CaBP 4），并与先天性静止性夜盲症有关。通过继续我们的密集 视网膜Ca 2+传感器蛋白的结构分析，并通过扩大范围， 膜运输调节因子（RD 3）和蛋白质靶点（RetGCs和离子通道），我们希望 获得视网膜钙传感器蛋白如何调节它们的原子水平的理解。 视网膜疾病过程中的膜结合靶蛋白。具体目标有三个方面：（1） 确定GCAP 1和RD 3各自与RetGC结合的原子级结构，以阐明激活 RetGCs的机制，从而为理解视觉的机制提供了结构基础。 （2）确定与CNG结合的CaM的结构 了解视杆和视锥光感受器光适应的分子机制的渠道; (3)确定视网膜钙传感器蛋白（CaBP 4）的原子水平结构， 视网膜L型电压门控Ca 2+通道（CaV1.4）在杆突触，以了解Ca 2 +- 与先天性静止性夜盲症相关的离子通道的依赖性调节机制。

The overall objectives are to develop nuclear magnetic resonance (NMR) techniques and use them in concert with experimental functional 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, xray 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) retinal guanylyl cyclase (RetGC) regulation by GCAP1 and retinal degeneration 3 (RD3) protein, and their role in autosomal dominant cone dystrophy and Leber Congenital Amaurosis; (2) Ca2+-dependent inactivation of photoreceptor cyclic nucleotide gated (CNG) channels controlled by calmodulin (CaM); and (3) Ca2+-dependent activation of retinal L-type Ca2+ channels (CaV1.4) mediated by calcium binding protein-4 (CaBP4), and implicated in congenital stationary night blindness. By continuing our intensive structural analysis of retinal Ca2+ sensor proteins and by broadening the scope to encompass membrane trafficking regulators (RD3) and protein targets (RetGCs and ion channels), we hope to gain an atomic-level understanding of how retinal calcium sensor proteins regulate their membrane-bound target proteins in retinal disease processes. The specific aims are 3-fold: (1) Determine atomic-level structures of GCAP1 and RD3 each bound to RetGC to elucidate activation mechanism of RetGCs and thus provide a structural basis for understanding mechanisms of visual recovery and retinal degenerative diseases; (2) Determine structures of CaM bound to CNG channels to understand molecular mechanisms of light-adaptation in rod and 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.