FOLDING AND ASSEMLY IN VISUAL RHODOPSIN

视觉视紫红质中的折叠和组装

• 批准号: 2430391
• 负责人: KEVIN Donald RIDGE
• 金额: $ 10.26万
• 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/2430391
• 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(摘自申请者的摘要)：蛋白质研究 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 光感受器视紫红质的集中鉴定和 牛视蛋白中独立折叠结构域的表征 载脂蛋白。遗传操作产生的视蛋白多肽片段 已经对牛视蛋白基因进行了体内功能组装的检测。 值得注意的是，两个或三个互补视蛋白多肽的共表达 分离在细胞质区域的碎片允许形成 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联合调节。为了进一步 证实这些发现，更多地点的本地化 视蛋白多肽链的不连续允许体内互补 正在被追查。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 碎片复合体的形成及其结构表征 圆二色谱(CD)和荧光光谱的性质 通过分析超速离心法确定它们的结合状态。按顺序 为进一步生产足够数量的视蛋白多肽片段 互补研究，细菌过表达正在进行中。这个 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 用滴定量热法进行检验。对展开的过渡的分析 差示扫描量热法测定视蛋白多肽片段 应验证它们是否由一个或多个独立的折叠组成 域名。除了识别包含牛视蛋白的区域之外 足够的信息独立折叠、插入到膜中，以及 组装成一个功能分子，这些研究应该提供洞察力 与一些自然发生的 视网膜色素变性患者的视蛋白突变。他们 预计还将与不断增长的折叠和组装相关 与鸟嘌呤核苷酸结合的相关受体数目 蛋白质。