FOLDING AND ASSEMLY IN VISUAL RHODOPSIN

视觉视紫红质中的折叠和组装

• 批准号: 2888476
• 负责人: KEVIN Donald RIDGE
• 金额: $ 10.36万
• 依托单位: UNIVERSITY OF MD BIOTECHNOLOGY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-06-01 至 2001-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2888476
• 关键词: Escherichia coli; SDS polyacrylamide gel electrophoresis; analytical ultracentrifugation; calorimetry; circular dichroism; cow; fluorescence spectrometry; gene complementation; genetic manipulation; protein biosynthesis; protein folding; protein reconstitution; receptor expression; rhodopsin; rod cell; site directed mutagenesis; thermodynamics; ultraviolet spectrometry; visual photoreceptor; western blottings

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.

描述（改编自申请人摘要）：蛋白质研究 折叠过程提供了深入了解的机制，动力学， 多肽折叠的热力学 这项研究的目的是 详细分析了杆状细胞的折叠和组装 感光视紫红质的重点鉴定和 牛视蛋白中独立折叠结构域的表征 脱辅基蛋白 基因操作产生的视蛋白多肽片段 的牛视蛋白基因的功能组装在体内进行了检查。 值得注意的是，共表达两个或三个互补的视蛋白多肽 在细胞质区域分离的片段允许形成 具有与天然色素相似的光谱特性的视紫红质。 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缔合介导。 进一步 为了证实这些发现，其他地点的本地化， 视蛋白多肽链的不连续性允许体内互补 正在被追捕 体外互补系统的开发将 通过检查促进视蛋白多肽 碎片复合物的形成和通过表征其结构 通过圆二色性（CD）和荧光光谱以及 作为它们的结合状态。 为了 以产生足够量的视蛋白多肽片段， 在互补研究中，细菌过表达正在进行中。 的 伴随片段互补的相互作用的性质和程度 自然突变和定点突变对这一过程的影响将 用滴定量热法检查。 展开转换的分析 通过差示扫描量热法在视蛋白多肽片段中 应验证它们是否由一个或多个独立折叠组成 域. 除了鉴定牛视蛋白中含有 足够的信息来独立折叠，插入膜中，和 组装成一个功能分子，这些研究应该提供见解 一些自然发生的 视网膜色素变性患者中的视蛋白突变。 他们 也有望与日益增长的 与鸟嘌呤核苷酸结合偶联的相关受体的数量 proteins.