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，我提议参加哥伦比亚大学生物化学和分子生物物理系杰出的x射线晶体学家Wayne a . Hendrickson的培训课程。本提案的具体目的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）、细胞视黄醛结合蛋白（CRALBP）和视紫红质。编码ATTP的基因突变已被证明可导致伴孤立性维生素E缺乏症的晚发性视网膜营养不良和共济失调，这是一种常染色体隐性遗传病，以进行性脊髓小脑变性为特征。CRALBP的突变已被证明可引起许多常染色体隐性视网膜营养不良症。最后，视紫红质的突变已被证明可引起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细胞质蛋白，并调节其对全身（维生素E）或光感受器（11-顺式视网膜）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。视紫红质是一种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质的高分辨率结构，但激活形式的视紫质结构仍然未知。我的目的是确定激活的视紫红质的结构与转导蛋白，其同源的G蛋白复合体。这样的结构将对整个GPCR家族产生影响，以及引起视紫红质突变的疾病。特异性目的1是表达和纯化CRALBP和ATTP。具体目标2是CRALBP和ATTP的结晶和结构测定。特异性目的3是测定天然牛视紫红质的结晶和结构。特异性目的4是单独或与转导蛋白一起测定活化的牛视紫红质的结晶和结构。Specific Aim 5是一种适合于Specific Aim 4和5的视紫红质表达系统的开发，视紫红质突变是追求视紫红质活性状态结构的一种替代策略。特异性目标6是使用目标3、4和5中描述的系统，通过突变体和x射线结构测定来分析视紫红质信号转导的机制。

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.