Folding and assembly in rhodopsin. Effect of mutations in the sixth transmembrane helix on the conformation of the third cytoplasmic loop.
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.
Center for Advanced Research in Biotechnology, National Institute of Standards and Technology and the University of Maryland Biotechnology Institute, Rockville, Maryland 20850, USA. ridge@carb.nist.gov
K D
KD
Ridge
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Ngo
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Lee
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Abdulaev
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003593:Cytoplasm / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D017510:Protein Folding; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D012243:Rhodopsin