An insertion or deletion in the extramembrane loop connecting helices E and F of archaerhodopsin-1 affects in vitro refolding and slows the photocycle.
Upon addition of retinal, archaeopsin-1 expressed in Escherichia coli (ecaO-1002) regenerated the chromophore in dimyristoyl phosphatidylcholine (DMPC), 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) and sodium dodecyl sulfate (SDS) mixed micelles as efficiently as the same opsin prepared from halobacteria. Introduction of an insertion or a deletion of five amino acids into the surface loop connecting helices E and F changed the secondary and tertiary structures of ecaO-1002 in SDS, and diminished regeneration of the chromophore. The effect of the insertion and deletion on the in vitro refolding was specific to archaeopsin because the same insertion introduced at the corresponding position of bacterioopsin (bO) did not affect chromophore regeneration. The photocycle of the regenerated ecaR-1002 decreased in DMPC/CHAPS/SDS mixed micelles compared with that of aR-1 in the claret membrane, which was consistent with the reported behavior of bO. Unexpectedly, the insertion and deletion in loop EF perturbed the photocycle of the regenerated ecaR-1002. The accumulation of long-lived N- and O-like intermediates suggested that the insertion and deletion slowed down the proton uptake steps at the cytoplasmic surface.
Sugiyama
Y
Y
Division of Biological Science, Nagoya University, Japan. Ysugiya@bio.nagoya-u.ac.jp
Koyanagi
T
T
Yamada
N
N
Mukohata
Y
Y
D000595:Amino Acid Sequence; D019843:Archaeal Proteins; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000528:radiation effects*; D004926:Escherichia coli / Q000235:genetics; D006217:Halobacterium / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010777:Photochemistry; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017494:Proton Pumps
D019843:Archaeal Proteins; D001426:Bacterial Proteins; C055476:archaerhodopsin protein, Archaea; D017494:Proton Pumps