CONFORMATIONAL CHANGES AND RHODOPSIN

描述（改编自申请人的摘要）：广泛，长期 拟议研究的目标是确定所使用的机制， 视觉受体视紫红质结合并激活G蛋白转导蛋白。 为了实现这一目标，构象的位置和时间 必须首先确定发生的变化。 随后， 可以解决这些改变中的哪一个是最低限度需要的。 视紫红质是 G蛋白偶联受体的最佳表征和理解。 因此，在本发明中， 本申请中提出的研究将提供更深入的理解， 这些受体如何被激活的结构水平。 拟议的研究将集中在观察到的具体构象变化 发生在视紫红质的跨膜螺旋上。 通过 采用一系列物理和生物化学方法来研究一系列 在细胞质中具有独特半胱氨酸残基的视紫红质突变体 在螺旋的末端，该建议将解决以下三个问题 问题：（1）是否可以检测到视紫红质的构象变化 使用荧光和生物化学技术;（2）关系是什么 这些构象变化与转导蛋白结合的位置之间的关系; 以及（3）是否需要构象变化来使视紫红质 以激活转导素。 后一个问题将使用 上述技术使用已知功能缺陷的视紫红质突变体 (i.e.,不能结合和激活转导素的蛋白质）。 这种突变体 型包括常染色体显性视网膜色素变性，一种 夜盲症 这些研究的结果可能有助于提供一个 从分子和机制上解释这种疾病， 关于信号转导机制中的主要步骤的一般知识 G蛋白偶联受体

DESCRIPTION (Adapted from applicant's abstract): The broad, long-term objectives of the proposed research are to determine the mechanism used by the visual receptor rhodopsin to bind and activate the G-protein transducin. To achieve this objective, the location and timing of the conformational changes that occur must first be determined. Subsequently, the question of which of these changes is minimally required can be addressed. Rhodopsin is the best characterized and understood G-protein coupled receptor. Thus, studies proposed in this application will provide a deeper understanding, at the structural level, of how these receptors become activated. The proposed research will focus on specific conformational changes observed to occur in the transmembrane helices of rhodopsin upon photoactivation. By employing a range of physical and biochemical approaches to study a series of rhodopsin mutants that have unique cysteine residues at the cytoplasmic ends of the helices, this proposal will address the following three questions: (1) whether conformational changes in rhodopsin can be detected using fluorescence and biochemical techniques; (2) what the relationship is between these conformational changes and the location of transducin binding; and (3) whether the conformational changes are required to enable rhodopsin to activate transducin. The latter question will be investigated using the above techniques with rhodopsin mutants known to be functionally defective (i.e., proteins that cannot bind and activate transducin). Mutants of this type include those found in autosomal dominant retinitis pigmentosa, a form of night blindness. The results from these studies may help to provide a molecular, mechanistic explanation for this disease, and increase our general knowledge about a primary step in the signal transduction mechanism of G-protein coupled receptors.