Reconstitution of bacteriorhodopsin from a mixture of a proteinase V8 fragment and two synthetic peptides.
The peptide and retinal mixture of bacteriorhodopsin, composed of two synthetic peptides corresponding to helices F (160-197) and G (202-237) and a proteinase V8-derived fragment V1 (1-166), generated the characteristic features of bacteriorhodopsin with absorbance maximum at 550 nm and fluorescence quenching as in two synthetic peptides corresponded to helix A (sequence 7-31) and B (41-65) and a chymotryptic fragment (72-248). The recovery of reconstitution estimated from the absorption and the fluorescence quenching of these mixture was 16-19% and 25-32% of the native purple membrane, respectively, whereas mixtures lacking any one of the peptides exhibited no absorption recovery Circular dichroism of each peptide fragment showed complete formation of alpha-helical structure in a membrane-mimetic medium of sodium dodecyl sulfate. These results indicate that the specific interactions or mutual recognitions between alpha-helices in lipid bilayers are essential for correct bundling of the seven helices and formation of the retinal binding pocket.
Ozawa
S
S
Department of Agricultural Chemistry, Faculty of Agriculture Kyoto University, Japan. ozawa@kais.kais.kyoto-u.ac.jp
Hayashi
R
R
Masuda
A
A
Iio
T
T
Takahashi
S
S
D000595:Amino Acid Sequence; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D003902:Detergents; D019617:Halobacterium salinarum / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017433:Protein Structure, Secondary; D012172:Retinaldehyde; D012697:Serine Endopeptidases; D013050:Spectrometry, Fluorescence
D003902:Detergents; D010446:Peptide Fragments; D001436:Bacteriorhodopsins; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D012172:Retinaldehyde