Folding and Assembly of Rhodopsin from Expressed Fragments

Published

March 1, 2000

Author(s)

K D. Ridge, N G. Abdulaev

Abstract

The dim-light photoreceptor rhodopsin is a prototypical member of the family of seven-transmembrane helix receptors that are coupled to guanine nucletide-binding proteins (G-proteins). Although chemical aspects of the membrane-embedded chromophore environment, the cytoplasmic sites of interaction for proteins involved in visual transduction and desensitization, and the intradiscal posttranslational modifications of rhodopsin have been the subject of active investigation, the relative contributions of these three regions to the overall folding and assembly of the photoreceptor have received considerably less attention. An understanding of how rhodopsin adopts it s tertiary structure is important not only to clarify details of the folding and assembly process, but also to gain insight into the severe visual impairments occurring as an immediate consequence of natural mutations affecting opsin structure and function. One approach that has been used successfully to study the mechanism of protein folding and assembly is to use fragments of a polypeptide. It is well known that certain polypeptide fragments behave as independent folding domains,^1-3 and membrane protein fragments have been functionally recombined in vitro and invivo.^4-11 The ability to form native-like rhodopsin complexes from expressed polypeptide fragments would provide opportunities for the study of a number of important aspects of bovine opsin folding and assembly. They could offer insights into which regions of the polypeptide chain contain sufficient information to fold independently, insert into a membrane, and assemble to form a retinal-binding opsin. They could also give information about the locations of topological determinants, interhelical interactions within the opsin apoprotein, and the minimum structure required for the binding of retinal. Of considerable interest is the prospect of examining the structural consequences of both site-directed and natural mutations on the folding and assembly of various segments of the opsin polypeptide chain. The purpose of this chapter is to describe methods for investigating whether expressed complementary bovine opsin fragments fold and assemble in vivo.^12-14 The focus is on the construction of opsin gene fragments, their transient expression in COS-1 cells, the biochemical characterization of the expressed fragments, and their implications to the overall mechanism of opsin biosynthesis.

Citation

Methods In Enzymology

Volume

315

Issue

Chap. 4

Pub Type

Journals

Keywords

complementation, membrane protein, protein folding, receptor, signal transduction, vision

Citation

Ridge, K. and Abdulaev, N. (2000), Folding and Assembly of Rhodopsin from Expressed Fragments, Methods In Enzymology (Accessed April 19, 2024)