Photoreceptor protein from the purple membrane of Halobacterium halobium. Molecular weight and retinal binding site.
The apparent molecular weight of the purple membrane protein of Halobacterium halobium was found to be 20 000 by sodium dodecyl sulfate gel electrophoresis and by gel filtration in sodium dodecyl sulfate. However, the molecular weight value determined by gel filtration in 6 M guanidine was 28 000. To resolve this discrepancy, methods insensitive to or independent of the conformation of the protein were used to estimate the molecular weight. Analytical ultracentrifugation of the sodium dodecyl sulfate-protein complex, peptide mapping, and amino acid analysis all gave values of 25 000 +/- 1000, a figure in agreement with a recent x-ray study. Borohydride reduction was used to attach the retinal cofactor covalently to a lysine residue. After digestion with thermolysin, peptide maps were prepared of the protein labeled at lysine residues with [14C] succinic anhydride both before and after reduction. Comparison of the maps showed one radioactive peptide with changed mobility. This peptide was isolated and shown to have the sequence Val-Ser-Asp-Pro-Asp-Lys-Lys with only one of the two lysine residues alkylated. Solid-phase sequencing showed the succinyl group to be at position 6 and hence the retinal group to be at position 7. It was possible that a small amount of retinal was also bound to Lys-6. There was no apparent homology with the corresponding peptide of vertebrate rhodopsin. No evidence of chain heterogeneity was found by radiochemical peptide mapping and sequence analysis of peptides containing lysine residues indicating that all protein chains of purple membrane are very similar or identical.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001426:Bacterial Proteins / Q000378:metabolism; D001665:Binding Sites; D006217:Halobacterium; D008566:Membranes; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D010786:Photoreceptor Cells; D011485:Protein Binding; D012172:Retinaldehyde / Q000378:metabolism*; D013820:Thermolysin; D014461:Ultracentrifugation; D014801:Vitamin A / Q000031:analogs & derivatives*
D000596:Amino Acids; D001426:Bacterial Proteins; D010446:Peptide Fragments; D014801:Vitamin A; D013820:Thermolysin; D012172:Retinaldehyde