STUDIES OF VISUAL CYCLE PROTEINS

视觉周期蛋白的研究

• 批准号: 3263014
• 负责人: JOHN W CRABB
• 金额: $ 18.22万
• 依托单位: ADIRONDACK BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-07-01 至 1989-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3263014
• 关键词: X ray crystallography; all trans retinol; autoradiography; binding proteins; biochemical evolution; chemical cleavage; cow; genetic library; high performance liquid chromatography; mass spectrometry; molecular cloning; nucleic acid sequence; nucleic acid structure; oligonucleotides; phosphorylation; protein sequence; protein structure; proteolysis; retinal pigment epithelium; retinaldehyde; transport proteins

The major emphasis of the proposed research is to elucidate the complete primary structure of the cellular retinaldehyde-binding protein (CRALBP) from bovine retina, a protein detected only in retina and retinal pigment epithelium (RPE) and which endogenously carries 11-cis-retinaldehyde and/or 11-cis-retinol. CRALBP could be a key component of the dark regeneration of 11-cis-retinaldehyde in the visual cycle and may also function as a substrate carrier protein for an 11-cis-retinol dehydrogenase found in RPE. Appropriate attention will be given to the identification of the NH2-terminal blocking group by fast atom bombardment-mass spectroscopy, to the identification possible sites of phosphorylation and to the evolutionary relatedness of CRALBP to other proteins. A concerted effort will also be made to crystallize CRALBP in a form suitable for X-ray crystallographic analysis. A long term goal is to obtain the three dimensional structure of the CRALBP 11-cis-retinaldehyde complex. The whole protein (size 33,000 daltons) will be fragmented by limited proteolysis to provide substructural domains which will be tested for residual retinoid-binding properties. The whole denatured protein will also be cleaved at asparaginyl-glycine, aspartyl-proline, methionyl, lysyl or arginyl bonds to generate fragments. The products of the various cleavage reactions will be purified and subjected to Edman degradation by automatic gas-phase and manual methodologies. Integration of these results is expected to provide the complete amino acid sequence. A coordinated approach will also be made to clone the gene for CRALBP and determine the cDNA structure. Met, Trp and Cys containing peptides will be selectively isolated and analyzed for their design potential for synthetic oligonucleotide hybridization probes. CRALBP antibodies will also be used for cDNA screening. Structural analysis will provide a precise molecular framework in which questions concerning the normal function of CRALBP and the visual disorders it may be associated with can be answered in specific terms.

拟议研究的主要重点是阐明完整 细胞性视网膜结合蛋白（CRALBP）的一级结构 来自牛视网膜，仅在视网膜和视网膜色素中检测到蛋白质 上皮（RPE），其内源性携带11-cis- retinaldenyde和/或 11-Cis-Retinol。 cralbp可能是黑暗再生的关键组成部分 在视觉循环中的11- cis-果仁醛，也可以用作 底物载体蛋白，用于发现在 RPE。 适当注意的是 NH2末端阻塞组通过快速原子轰击质量光谱法， 鉴定磷酸化的位置和 Cralbp与其他蛋白质的进化相关性。 一致的努力 还将制定以适合X射线的形式结晶cralbp 晶体学分析。 一个长期目标是获得三个 Cralbp 11-Cis- retinaldenaldehyde复合物的尺寸结构。 整个蛋白质（33,000 daltons尺寸）将被限制分散 蛋白水解提供将测试的子结构域 残留的类维生素性结合特性。 整个变性蛋白将 也可以在天冬酰胺基甘氨酸，天冬氨酸，甲基，赖氨酸上切割 或精氨酸键产生片段。 各种产品 切割反应将被纯化，并通过Edman降解。 自动气相和手动方法。 这些结果的整合 预计将提供完整的氨基酸序列。 协调 还将采用方法来克隆Cralbp的基因并确定 cDNA结构。 Met，含有肽的TRP和CYS将有选择性 隔离和分析其合成的设计潜力 寡核苷酸杂交探针。 也将使用CRALBP抗体 用于cDNA筛选。 结构分析将提供精确的分子 关于Cralbp和Cralbp正常功能的问题的框架和 它可能与之相关的视觉障碍可以用特定的回答 术语。