FOLDING AND ASSEMLY IN VISUAL RHODOPSIN

视觉视紫红质中的折叠和组装

• 批准号: 2711140
• 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/2711140
• 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）：蛋白质的研究