Membrane targeting calcium sensors in vision

总的目标是开发核磁共振技术和用途 与实验功能方法相结合，以阐明分子结构和 参与光信号转导的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 核磁共振、荧光、微量热、自旋标记电子顺磁共振、X射线结晶学、 和计算分析来描绘一族的结构、动力学和机制 神经元钙感受器蛋白(钙-肉豆蔻酸基开关)，作为膜- 靶向钙信号调节因子，并与视网膜和神经疾病有关。我们的 研究将确定以下结构基础：(1)视网膜鸟苷酸环化酶(RetGC)通过以下方式调节 GCAP1和视网膜变性3(RD3)蛋白及其在常染色体显性视锥中的作用 营养不良与Leber先天性巨结肠；(2)钙依赖的光感受器失活 钙调素(CaM)控制的环核苷酸门控(CNG)通道；(3)钙依赖 钙结合蛋白-4介导的视网膜L型钙通道的激活 (CaBP4)，并与先天性静止性夜盲有关。通过继续我们的密集 视网膜钙离子感受器蛋白的结构分析及扩大研究范围 膜转运调节因子(RD3)和蛋白质靶标(RetGCs和离子通道)，我们希望 在原子水平上了解视网膜钙感受器蛋白是如何调节它们的 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) 测定与RetGC结合的GCAP1和RD3的原子级结构以阐明激活 RetGCs的机制，从而为理解视觉机制提供了结构基础 康复与视网膜退行性疾病；(2)确定与CNG结合的CaM的结构 了解视杆细胞和视锥细胞光适应的分子机制； (3)测定视网膜钙感受器蛋白(CaBP4)结合的原子级结构 视杆突触视网膜L型电压门控性钙通道的研究 离子通道依赖的调节机制与先天性静止性夜盲有关。

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.