Abstract
Previous studies on bovine opsin folding and assembly have identified an amino-terminal fragment, EF(1-232), which folds and inserts into a membrane only after coexpression with its complementary carboxyl-terminal fragment, EF(233-348). To further characterize this interaction, EF(1-232) production was examined upon coexpression with carboxyl-terminal fragments of varying length and/or amino acid composition. These included fragments with incremental deletions of the third cytoplasmic loop (TH(241-348) and EF(249-348)), a fragment composed of the third cytoplasmic loop and sixth transmembrane helix (HF(233-280)), a fragment composed of the sixth and seventh transmembrane helices (FG(249-312)), and EF(233-348) and TH(241-348) fragments with Pro-267 or Trp-265 mutations. Although EF(1-232) production was independent of the third cytoplasmic loop and carboxyl-terminal tail, both the sixth and seventh transmembrane helices were essential. The effects of mutations in the sixth transmembrane helix on EF(1-232) expression were dependent on the length of the third cytoplasmic loop. Although Pro-267 mutations in EF(233-348) failed to stabilize EF(1-232) expression, their introduction into TH(241-348) was without discernible effects. However, Trp-265 substitutions in the EF(233-348) and TH(241-348) fragments conferred significant EF(1-232) production. Therefore, key residues in the transmembrane helices may exert their effects on opsin folding, assembly, and/or function by influencing the conformation of the connecting loops.
Highlights
Previous studies on bovine opsin folding and assembly have identified an amino-terminal fragment, EF[1– 232], which folds and inserts into a membrane only after coexpression with its complementary carboxyl-terminal fragment, EF[233–348]
Bovine rhodopsin is composed of the apoprotein opsin, a single polypeptide chain of 348 amino acids, and an 11-cisretinal chromophore [1,2,3,4]
The apoprotein folds into a structure of seven transmembrane (TM)1 helices connected by solvent exposed polypeptide segments on the intradiscal and cytoplasmic surfaces
Summary
Previous studies on bovine opsin folding and assembly have identified an amino-terminal fragment, EF[1– 232], which folds and inserts into a membrane only after coexpression with its complementary carboxyl-terminal fragment, EF[233–348]. All of the singly expressed fragments fold to a conformation that allows for membrane insertion and, in some cases, form the rhodopsin chromophore with 11-cis-retinal when coexpressed with their complementary partners These results suggest that the functional assembly of bovine rhodopsin is mediated by the association of multiple folding domains and demonstrate the utility of defined polypeptide fragments for studying the mechanism of bovine opsin folding and assembly. Coupled with our earlier findings, the present results suggest that specific amino acid residues in the TM helices can exert their effects on opsin folding, membrane insertion, and/or assembly by influencing the conformation of the solvent exposed connecting loop regions
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
