X-ray Structural Studies of Retinal Dystrophies

描述（由申请人提供）： 我的目标是利用x射线晶体学研究视网膜和神经系统疾病的结构基础。为了成为结构生物学领域的独立研究者，我打算接受杰出的X射线晶体学家韦恩A。哥伦比亚大学生物化学与分子生物物理系。该提案的具体目标涉及与视网膜营养不良相关的三种蛋白质的结构测定：α-生育酚转移蛋白（ATTP）、细胞视黄醇结合蛋白（CRALBP）和视紫红质。编码ATTP的基因中的突变已显示引起晚发性视网膜营养不良和共济失调伴孤立性维生素E缺乏，这是一种以进行性脊髓小脑变性为特征的常染色体隐性病症。CRALBP的突变已被证明会导致许多常染色体隐性视网膜营养不良。最后，视紫红质的突变已被证明会导致常染色体显性视网膜色素变性（ADRP）和先天性静止性夜盲症。ATTP和CRALBP都是结合不溶性疏水小分子的胞质蛋白，并调节它们对整个身体（对于维生素E）或光感受器（对于11-顺式-视黄醇）的可用性。这些蛋白质的结构应该有助于阐明这类蛋白质的功能。视紫红质是G蛋白偶联受体（GPCR），可能是最重要的一类受体分子。虽然最近报道了基态视紫红质的高分辨率结构，但视紫红质的活化形式的结构仍然是未知的。我的目的是确定结构的激活视紫红质的复合物与transducin，其同源G蛋白。这种结构将对整个GPCR家族产生影响，以及导致视紫红质突变的疾病。具体目标1是CRALBP和ATTP的表达和纯化。具体目标2是CRALBP和ATTP的结晶和结构测定。具体目标3是天然牛视紫红质的结晶和结构测定。具体目标4是单独或与转导蛋白一起的活化的牛视紫红质的结晶和结构测定。特定目标5是开发适用于特定目标4和5的视紫红质表达系统，视紫红质突变体作为追求视紫红质活性状态结构的替代策略。具体目标6是使用目标3、4和5中描述的系统，使用突变体和X射线结构测定来分析视紫红质的信号转导机制。

DESCRIPTION (provided by applicant): My goal is to investigate the structural basis of retinal and neurological disorders using x-ray crystallography. In order to become an independent investigator in the field of structural biology, I propose to undertake a course of training with preeminent x-ray crystallographer, Wayne A. Hendrickson, Department of Biochemistry and Molecular Biophysics, Columbia University. The specific aims of this proposal concern the structural determination of three proteins that are related to retinal dystrophies: alpha-tocopherol transfer protein (ATTP), cellular retinaldehyde binding protein (CRALBP), and rhodopsin. Mutations in the gene encoding ATTP have been shown to cause a late onset retinal dystrophy and ataxia with isolated vitamin E deficiency, an autosomal recessive condition characterized by a progressive spinocerebellar degeneration. Mutations in CRALBP have been shown to cause a number of autosomal recessive retinal dystrophies. Finally, mutations in rhodopsin have been shown to cause autosomal dominant retinitis pigmentosa (ADRP) and congenital stationary night blindness. ATTP and CRALBP are both cytosolic proteins that bind insoluble hydrophobic small molecules, and regulate their availability to the whole body (for vitamin E) or the photoreceptor (for 11-cis-retinal). The structures of these proteins should help to elucidate the function of this class of proteins. Rhodopsin is a G protein coupled receptor (GPCR), perhaps the most important class of receptor molecules. Although a high resolution structure of ground-state rhodopsin was recently reported, the structure of the activated form of rhodopsin is still unknown. My aim is to determine the structure of activated rhodopsin in a complex with transducin, its cognate G protein. Such a structure would have ramifications for the entire GPCR family, as well as disease causing mutations of rhodopsin. Specific Aim 1 is expression and purification of CRALBP and ATTP. Specific Aim 2 is crystallization and structure determination of CRALBP and ATTP. Specific Aim 3 is crystallization and structural determination of native bovine rhodopsin. Specific Aim 4 is crystallization and structure determination of activated bovine rhodopsin alone or with transducin. Specific Aim 5 is development of an expression system for rhodopsin suitable for use in Specific Aim 4 and 5, with mutants of rhodopsin as an alternative strategy to pursue the structure of the active state of rhodopsin. Specific Aim 6 is the use of the systems described in Aims 3, 4, and 5, to analyze the mechanism of signal transduction by rhodopsin using mutants and x-ray structure determination.