LONG-RANGE EFFECTS ON THE RETINAL CHROMOPHORE OF BACTERIORHODOPSIN CAUSED BY SURFACE CARBOXYL GROUP MODIFICATION

Carboxyl groups of bacteriorhodopsin (bR) that are modified by 1-ethyl -3-[3-(trimethylamino)propyl]carbodiimide (ETC) have been identified. Reaction of deionized purple membrane with a 400-fold molar excess of ETC or [C-14]ETC for 1 h at 0 degrees C incorporates about 3.5 mol of ETC/mol of bR. Proteinase K cleavage of ETC-modified bacterioopsin (bO ) produced small C-14-labeled peptides. Amino acid sequence analysis s howed three major ETC-modified residues: Glu 234, Asp 38, and Glu 74. Proteolysis of purple membrane with papain removes the ETC site at Glu 234. Treatment of ETC-modified, papain-cleaved purple membrane with h ydroxylamine removes half of the remaining ETC label. Subsequent cleav age with chymotrypsin, followed by amino acid sequence analysis, revea led that most of the remaining label was at Glu 74. bR modified by ETC primarily at Glu 74 displays two alterations in the retinal chromopho re, located in the membrane interior at a distance more than 2 nm away from the modified carboxyl group. (1) The acid-induced purple-to-blue transition undergoes a shift in apparent pK from 3.2 to 2.3. (2) The second-order rate constant for chromophore regeneration from bO and re tinal is diminished from 3600 to 1700 M(-1) s(-1) in membrane sheets. Most of the shift in the pK of the purple-to-blue transition can be ex plained by the quaternary ammonium ion of ETC attached to Glu 74 overl apping the postulated location of the guanidinium group of Arg 82. The inhibition of regeneration may be attributed to the involvement of th e loop connecting helices B and C in a conformational change between b O and bR.