VISUAL CYCLE PROTEINS

细胞视黄醇结合蛋白的结构与功能研究 （CRALBP）提出了专注于功能结构域分析， 蛋白质制剂的开发适用于三维 结构分析该提案的假设是，CRALBP发挥 在视觉周期中维生素A的代谢中起着重要作用。 CRALBP是仅在视觉组织中发现的底物载体蛋白， 携带11-顺式-视黄醇和11-顺式-视黄醇醛， 在视觉中起作用这种蛋白质似乎在调节 作用，影响11-顺式-视黄醇是否作为视黄酯储存在 视网膜色素上皮（RPE）或氧化成11-顺式-视黄醇， 输出用于感光细胞中的视色素再生。cralbp 在体外刺激氧化和阻碍酯化，表明， 该蛋白质可以帮助控制关键代谢分支点类维生素A流动 在视觉周期中。合成、调节或结构缺陷 CRALBP可能会损害维生素A代谢，导致受损 视野 功能结构域分析将利用人重组CRALBP 并应用酰胺氢/氘交换和质谱法， 拓扑分析，以便于识别隐藏域 与类维生素A结合相关， 蛋白质相互作用 rCRALBP的光亲和标记将是 进行鉴定类维生素A结合口袋和交联的 通过Edman降解和电喷雾质谱法确定的位点。 定点诱变的靶点将基于来自 这些方法和特定氨基酸的变化用于定义 类维生素A结合和与11-顺式-视黄醇相互作用的关键残基 脱氢酶。 有限的丙氨酸扫描诱变也将集中在 在一个随机的rCRALBP突变体中， 结合类维生素A将开发一种程序， rCRALBP用于三维结构测定的长期目标， 结晶学 有限的蛋白水解将用于鉴定 能够结合类维生素A的最小片段。类维生素A结合 片段也将作为用于溶液的重组蛋白产生 NMR结构分析。蛋白水解的底物载体效能 片段和突变体将使用牛RPE微粒体进行测定， 11-顺式-视黄醇脱氢酶来源。出于比较的目的， 牛GRALBP将用作结构-功能参考。的统一 这项研究的假设是，通过建立分子基础， 了解CRALBP的正常结构、功能和调节， 关于蛋白质可能与视觉障碍有关的问题 可以用具体的术语来回答。

Structure-function studies of the cellular retinaldehyde-binding protein (CRALBP) are proposed that focus upon functional domain analysis and development of protein preparations suitable for three dimensional structural analysis. The hypothesis of the proposal is that CRALBP plays a fundamental role in the metabolism of vitamin A in the visual cycle. CRALBP is a substrate carrier protein found only in visual tissue that carries 11-cis-retinol and 11-cis-retinaldehyde, retinoids that are only known to function in vision. The protein appears to play a regulatory role, influencing whether 11-cis-retinol is stored as retinyl ester in the retinal pigment epithelium (RPE) or oxidized to 11-cis-retinaldehyde and exported for visual pigment regeneration in photoreceptor cells. CRALBP stimulates oxidation and retards esterification in vitro, suggesting that the protein may help control retinoid flow at a key metabolic branch point in the visual cycle. A defect in the synthesis, regulation or structure of CRALBP could compromise vitamin A metabolism, resulting in impaired vision. Functional domain analysis will utilize human recombinant CRALBP and apply amide hydrogen/deuterium exchange and mass spectrometry for topological analysis to facilitate identification of buried domains associated with retinoid-binding and exposed regions involved with protein-protein interactions. Photoaffinity-labeling of rCRALBP will be performed to identify the retinoid-binding pocket and the crosslinked sites determined by Edman degradation and electrospray mass spectrometry. Targets for site-directed mutagenesis will be based upon results from these approaches and changes in specific amino acids used to define residues critical for retinoid-binding and interaction with 11-cis-retinol dehydrogenase. Limited alanine scanning mutagenesis will also focus around two residues in a random rCRALBP mutant that has lost the ability to bind retinoid. A procedure will be developed for crystallizing human rCRALBP for the long term goal of 3D structural determination by crystallography. Limited proteolysis will be used to identify the smallest fragment capable of binding retinoid. The retinoid-binding fragment will also be produced as a recombinant protein for solution structural analysis by NMR. The substrate carrier efficacy of proteolysis fragments and mutants will be assayed using bovine RPE microsomes as the source of 11-cis-retinol dehydrogenase. For comparative purposes, native bovine GRALBP will be used as a structure-function reference. The unifying hypothesis of the research is that by establishing a molecular basis for understanding the normal structure, function and regulation of CRALBP, questions concerning visual disorders with which the protein may be associated can be answered in specific terms.