EFFECT OF MUTATIONS ON RHODOPSIN STRUCTURE AND FUNCTION

突变对视紫红质结构和功能的影响

• 批准号: 3267053
• 负责人: Sadashiva S Karnik
• 金额: $ 15.57万
• 依托单位: CLEVELAND CLINIC FOUNDATION
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-04-01 至 1997-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3267053
• 关键词: clone cells; disease /disorder model; disulfide bond; gene mutation; molecular pathology; protein folding; protein structure function; radiotracer; retinitis pigmentosa; rhodopsin; rod cell; synthetic nucleic acid; transducin; transfection; visual phototransduction

A unique disulfide bond between C110 and C187 is critical for rhodopsin structure. Mutants of intradiscal loops, and some of the autosomal dominant retinitis pigmentosa (ADRP) mutants are believed to be defective in folding resulting in the lack of S-S bond formation. Our long term goal is to understand structure-function relationship in bovine rhodopsin by expression in COS cells and characterization of the mutant proteins. This proposal is aimed at: 1. investigating the molecular basis of defects caused by the absence of the S-S bond. 2. identifying Light induced changes in transmembrane helices C and F of opsin. We will examine directly in the membranes, the ability of mutants C110S & C187S to bind the chromophore 11 -cis-retinal, by UV/Visible difference spectroscopy and by crosslinking of the 3H-retinal to opsin. We will examine whether or not a noncovalent interaction between residues 110-187 suppresses the defect. 1-3 nmoles of mutant proteins can be obtained from transfected COS cells. Retinal binding to wildtype and unglycosylated opsin can be measured by spectroscopy and crosslinking techniques. In a mutant, CllOE-Cl87R, a blue shifted chromophore with low yield was observed possibly due to an ion pair interaction of the introduced residues. A systematic study of ion pair replacement of the S-S bond will help us establish the mechanism by which stabilization of the retinal pocket takes place. To identify the defect in some of the ADRP mutations, substitution of amino acids with different side chain characters will be made at seven ADRP sites. The location, post-translational modification and the function of the mutant proteins expressed in COS cells will be examined. The number of S-S bonds in the expressed defective proteins will be measured. We have optimized a sensitive technique to measure the number of S-S bonds in opsin, based on the specific cleavage of disulfide bond by stoichiometric incorporation of 14CN. The wild type opsin from the COS cells and the ROS, incorporate lmol/mol CN. The defective mutants are expected to incorporate substoichiometric or none, if a defect in S-S bond formation indeed exists in them. The out come of this test will help us to understand the molecular basis of a heterogeneous degenerative neuronal disorder. Molecular modelling studies show that transmembrane helices C and F of rhodopsin interact closely with retinal, and therefore might be primarily responsible for transduction of signal across the membrane. To examine this we will create mutants with-two amino acid insertions in helices C & F and examine their ability to activate transducin in response to light. Contact of retinal with specific residues is expected to be disrupted by insertion and result in the loss of transducing ability. In another approach we will introduce cysteines at the cytoplasmic border of helices C & F. The solvent accessibility of the introduced cysteines in dark and light activated mutant proteins will be studied. This will be important to explain transduction in terms of intramolecular changes.

C110和C187之间的独特二硫键对视紫红质至关重要 结构 椎间盘内袢突变，以及一些常染色体 显性视网膜色素变性（ADRP）突变体被认为是有缺陷的 在折叠中导致缺乏S-S键形成。 我们的长期 目的是了解牛视紫红质的结构与功能关系 通过在COS细胞中的表达和突变蛋白的表征。 本建议旨在：1.研究的分子基础 由于缺少S-S键而导致的缺陷。2.识别灯 引起视蛋白跨膜螺旋C和F的变化。 我们将 直接在细胞膜上检测，突变体C110 S和C187 S 通过UV/可见光差结合发色团11 -顺式-视黄醛 光谱法和通过3 H-视黄醛与视蛋白的交联。 我们将 检查残基110-187之间的非共价相互作用是否 抑制缺陷。1-3 nmol的突变蛋白可以从 转染COS细胞。 与野生型和非糖基化的视网膜结合 视蛋白可以通过光谱学和交联技术测量。 中 突变体Cl 10 E-Cl 87 R是一个蓝移的低产量发色团， 观察到可能是由于引入的离子对相互作用 残基 S-S键离子对置换的系统研究 将有助于我们建立一种机制， 视网膜袋形成。 为了识别某些ADRP中的缺陷， 突变，用不同侧链取代氨基酸 将在七个ADRP站点制作字符。 地点， 翻译后修饰和突变蛋白的功能 将检测COS细胞中表达的蛋白。 分子中S-S键的数目 将测量表达的缺陷蛋白。 我们优化了 一种灵敏的技术来测量视蛋白中S-S键的数量，基于 通过化学计量掺入特异性切割二硫键 14CN。 来自COS细胞的野生型视蛋白和ROS， lmol/mol CN。 这些缺陷突变体有望 亚化学计量或无，如果S-S键形成中的缺陷确实 存在于其中。 这个测试的结果将帮助我们理解 异质性变性神经元疾病的分子基础。 分子模拟研究表明， 视紫红质与视网膜密切相互作用，因此可能主要是 负责跨膜传递信号。 审查 这样我们就可以在C螺旋上插入两个氨基酸， 并检测它们在光刺激下激活转导素的能力。 预计视网膜与特定残留物的接触将被破坏， 插入并导致转导能力的丧失。 在另一 我们将在螺旋的细胞质边缘引入半胱氨酸 C & F。引入的半胱氨酸在黑暗中的溶剂可及性和 将研究光激活突变蛋白。 这很重要 用分子内的变化来解释转导。