IRBP--STRUCTURE/FUNCTION

描述(改编自申请者摘要)：互光感受器 维甲酸结合蛋白(IRBP)转运11-顺式和全反式维甲酸 RPE和光感受器之间通过溶解维甲酸和增强 11顺式视网膜从RPE中释放。我们的长期目标是理解 IRBP完成这些任务的机制。IRBP由四个同源序列组成 每个模块都包含两个疏水配体结合位点和一个 非配体结合区，是一个候选的对接位置 光感受器基质或细胞表面受体。根据推论，通过 基因复制模块的结构不同，创建了量身定做的位置 对于IRBP的每一种生理配体。工作假说是 通过基因复制和分化，这些模块获得了结合位点 为不同的配体以及与基质相互作用的结构域量身定做 或者细胞表面。提出了三个具体目标：(1)假设预测 IRBP拥有两类模块，每个模块优先绑定 不同的配位体。每个模块的配基亲和力和特异性将 与全长IRBP和选定的模块组合进行比较。 这将通过对单个模块的诱变研究来补充。两个人 模块内的结合位点将通过Arg到Gln和 Trp到Phe的取代。(2)IRBP功能的结构要求 将在生理条件下确定。使用眼部护理制剂， IRBP、各个模块、选定的模块组合和截断 将评估模块支持视紫红质再生的能力，以及 视黄醇在RPE根尖表面的传递和释放。完全生理学的 IRBP的活性可能需要模块和非配体结合的组合 可能是受体对接地点的区域。(3)X射线晶体结构 将确定单个IRBP模块的大小。单个IRBP的大小 模块是IRBP的功能单元，是X射线结晶学的理想选择。 初步研究表明，IRBP模块2的晶体将是 足够的质量来解决单个模块的结构问题。真知灼见 从晶体结构中获得的结果将为进一步的功能研究提供参考 未来的工作。

DESCRIPTION (Adapted from applicant's abstract): Interphotoreceptor retinoid-binding protein (IRBP) transports 11-cis and all-trans retinoids between the RPE and photoreceptors by solubilizing retinoids and enhancing the release of 11-cis retinal from the RPE. The long-term goal is to understand the mechanisms by which IRBP fulfills these tasks. IRBP consists of four homologous modules each containing two hydrophobic ligand-binding sites and a non-ligand-binding region that is a candidate docking site for an interphotoreceptor matrix or cell surface receptor. It is reasoned that through gene duplication the structure of the modules diverged, creating sites tailored for each of IRBP's physiological ligands. The working hypothesis is that through gene duplication and divergence the modules obtained binding sites tailored for different ligands as well as domains that interact with the matrix or cell surface. Three specific aims are proposed: (1) The hypothesis predicts that IRBP possesses two classes of modules, each preferentially binding different ligand sets. The ligand affinity and specificity of each module will be compared with that of the full-length IRBP and selected module combinations. This will be complemented by mutagenesis studies of a single module. The two binding sites within the module will be characterized through Arg to Gln and Trp to Phe substitutions. (2) The structural requirements for IRBP's function will be determined under physiological conditions. Using eyecup preparations, IRBP, the individual modules, selected module combinations and truncated modules will be assessed for ability to support rhodopsin regeneration, and the delivery and release of retinoids at the RPE apical surface. Full physiological activity of IRBP may require combinations of modules, and non-ligand-binding regions that could be receptor-docking sites. (3) The X-ray crystal structure of an individual IRBP module will be determined. The size of an individual IRBP module, the functional unit of IRBP, is ideal for X-ray crystallography. Preliminary studies indicate that crystals of IRBP module 2 will be of sufficient quality to solve the structure of an individual module. Insights gained from the crystal structure will suggest further functional studies for future work.