Removal of the carboxyl-terminal peptide does not affect refolding or function of bacteriorhodopsin as a light-dependent proton pump.
Treatment of the purple membrane with carboxypeptidase A, Pronase, or papain, results in the cleavage of amino acids from the carboxyl terminus of bacteriorhodopsin, a maximum of about 17 amino acids being released with papain. Protease-treated bacteriorhodopsin, after denaturation, refolds to the native structure, binds retinal as tightly as the intact protein and, on reconstitution into vesicles, gives full proton translocating activity. The CD spectrum of papain-treated purple membrane shows exciton coupling characteristic of the intact purple membrane. The trimeric bacteriorhodopsin in papain-treated purple membrane dissociates into monomers in Triton X-100 which, after removal of the detergent, reassociate to form the oligomeric structures. Chymotrypsin cleaves papain-treated bacteriorhodopsin between amino acids 71 and 72 as has been previously found for intact bacteriorhodopsin. The resulting fragments C-1 (amino acids 72-231) and C-2 (amino acids 1-71) reassociate, bind retinal, and regenerate the native chromophore, as previously demonstrated for the corresponding fragments from the intact protein. We conclude that the COOH-terminal peptide in bacteriorhodopsin is not required for the correct refolding of denatured bacteriorhodopsin to the native tertiary and quarternary structure, for chromophore regeneration or for light-driven proton translocation.
D000595:Amino Acid Sequence; D001436:Bacteriorhodopsins / Q000378:metabolism*; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D002338:Carotenoids / Q000378:metabolism*; D002918:Chymotrypsin; D002942:Circular Dichroism; D006217:Halobacterium / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008027:Light; D010446:Peptide Fragments / Q000378:metabolism; D011402:Pronase; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D012172:Retinaldehyde / Q000378:metabolism
D010446:Peptide Fragments; D002338:Carotenoids; D001436:Bacteriorhodopsins; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D002918:Chymotrypsin; D011402:Pronase; D012172:Retinaldehyde