STRUCTURE/FUNCTION STUDIES OF RHODOPSIN

这些研究的长期目标是解开分子机制。 视觉色素视紫红质的作用。这项工作将导致 设计特定的、活性部位定向的和/或基于机理的试剂 用于治疗视网膜色素变性和先天性视网膜病变等疾病 静止性夜盲。 这项建议有两个具体目的：(一)确定和 阐明必需氨基酸残基在视紫红质中的作用，以及(Ii) 利用这些信息设计活性部位定向试剂 目标是突变的视紫红质。 实验方法将是使用定点突变。 结合广泛的体外功能分析和化学 改变后的视紫红质的修饰。诱变技术将用于 确定激活和失活过程中的必需氨基酸 视紫红质和视黄素。将使用交联性研究来确定如何 紧密相连的是活性部位的必需残基。换能器 将进行活动分析，以确定被激活的 突变体类似于光激活的视紫红质。最后，增加了 了解野生型和突变型视紫红质的机制将是 用于设计基于视网膜类似物的活性部位定向试剂 特异性靶向视紫红质在视网膜炎患者中的发现 色素变性和先天性静止性夜盲。

The long term goal of these studies is to unravel the molecular mechanism of action of the visual pigment rhodopsin. This work will lead to the design of specific, active-site directed, and/or mechanism-based reagents aimed at treatment of diseases such as retinitis pigmentosa and congenital stationary night blindness. There are two specific aims in this proposal: (i) to identify and elucidate the role of essential amino acid residues in rhodopsin, and (ii) to use this information to design active-site directed reagents that target mutant rhodopsins. The experimental approach will be to use site-directed mutagenesis combined with extensive in vitro functional analysis and chemical modification of the altered rhodopsins. Mutagenesis will be used to identify essential amino acids in the activation and inactivation of rhodopsin and opsin. Crosslinking studies will be used to determine how close together are essential residues in the active site. Transducin activity assays will be performed to determine how closely the activated mutants resemble photoactivated rhodopsin. Finally, the increased understanding of mechanism in the wild-type and mutant rhodopsins will be used to design active-site directed reagents based on retinal analogs that specifically target mutant rhodopsins found in patients with retinitis pigmentosa and congenital stationary night blindness.