The site of attachment of retinal in bacteriorhodopsin. A resonance Raman study

The retinal chromophore of bacteriorhodopsin is attached as a Schiff's base with the epsilon-amino group of a lysine residue. The site of attachment has now been investigated by the use of resonance Raman spectroscopy which has previously been shown to be sensitive to 15N isotope substitution at the Schiff's base. Bacteriorhodopsin samples obtained from bacteria grown in a medium containing either [epsilon-14N]- or [epsilon-15N]lysine were cleaved with chymotrypsin to give, in each case, the two fragments C-1 (amino acids 72-248) and C-2 (amino acids 1-71). The fragments were recombined in different combinations into lipid/detergent mixtures and retinal was added to regenerate the chromophore. Resonance Raman spectroscopy showed that, in both the light-adapted (BR 570) and the M 412 intermediate forms, the chromophore is attached to the large C-1 fragment. This result eliminates Lys-41 as the attachment site in these forms of bacteriorhodopsin. Together with the accompanying report, which demonstrates that the epsilon-amino group in Lys-41 is not required for regeneration of the native chromophore or for proton translocation, these results provide strong evidence that the chromophore remains attached as a Schiff's base to Lys-216 during the entire photocycle.