FOLDING AND ASSEMLY IN VISUAL RHODOPSIN

描述（改编自申请人的摘要）：蛋白质的研究 折叠过程提供了对机制、动力学和 多肽折叠的热力学。 这项研究的目标是 提供杆状电池折叠和组装的详细分析 光感受器视紫红质通过专注于识别和 牛视蛋白独立折叠域的表征 脱辅基蛋白。 通过基因操作产生的视蛋白多肽片段 已对牛视蛋白基因的体内功能组装进行了检查。 值得注意的是，两个或三个互补视蛋白多肽的共表达 在细胞质区域分离的片段允许形成 视紫红质具有与天然色素相似的光谱特征。 这些结果表明视紫红质的功能组装可能是 由多个蛋白质折叠结构域的联合介导。 为了进一步 证实这些发现，其他网站的本地化 视蛋白多肽链的不连续性允许体内互补 正在被追捕。 体外补充系统的开发将 通过检查促进视蛋白多肽的条件来促进 片段复合物的形成并通过其结构表征 圆二色性 (CD) 和荧光光谱的特性 通过分析超速离心作为它们的结合状态。 按顺序 产生足够量的视蛋白多肽片段用于进一步 互补研究正在进行中，细菌过度表达。 的 伴随片段互补的相互作用的性质和程度 自然和定点突变对该过程的影响将 通过滴定量热法进行检查。 分析正在发生的转变 通过差示扫描量热法观察视蛋白多肽片段 应验证它们是否由一个或多个独立折叠组成 域。 除了识别含有牛视蛋白的区域外 足够的信息来独立折叠、插入膜中，以及 组装成功能分子，这些研究应该提供见解 与一些自然发生的结构后果相关 在患有色素性视网膜炎的患者中观察到视蛋白突变。 他们 预计也与不断增长的折叠和组装有关 与鸟嘌呤核苷酸结合偶联的相关受体的数量 蛋白质。

DESCRIPTION (Adapted from applicant's abstract): Studies of the protein folding process offer insights into the mechanisms, kinetics, and thermodynamics of polypeptide folding. The goal of this research is to provide a detailed analysis of the folding and assembly of the rod cell photoreceptor rhodopsin by focusing on the identification and characterization of independent folding domains in the bovine opsin apoprotein. Opsin polypeptide fragments generated by genetic manipulation of the bovine opsin gene have been examined for functional assembly in vivo. Remarkably, co-expression of two or three complementary opsin polypeptide fragments separated in the cytoplasmic region allows the formation of rhodopsins with spectral characteristics similar to the native pigment. These results suggest that the functional assembly of rhodopsin may be mediated by the association of multiple protein-folding domains. To further substantiate these findings, the localization of additional sites where discontinuity of the opsin polypeptide chain allows in vivo complementation is being pursued. The development of an in vitro complementing system will be facilitated by examining conditions which promote opsin polypeptide fragment complex formation and through characterization of their structural properties by circular dichroism (CD) and fluorescence spectroscopy as well as their state of association by analytical ultracentrifugation. In order to produce sufficient quantities of opsin polypeptide fragments for further complementation studies, bacterial overexpression is in progress. The nature and extent of the interactions accompanying fragment complementation and the effects of natural and site-directed mutations on the process will be examined by titration calorimetry. Analysis of the unfolding transitions in the opsin polypeptide fragments by differential scanning calorimetry should verify whether they are composed of one or more independent folding domains. In addition to identifying regions of bovine opsin which contain sufficient information to independently fold, insert into a membrane, and assemble into a functional molecule, these studies should provide insights into the structural consequences associated with some naturally-occurring opsin mutations seen in patients afflicted with retinitis pigmentosa. They are also expected to have relevance to the folding and assembly of a growing number of related receptors which are coupled to guanine nucleotide-binding proteins.