The chromophore retinal in bacteriorhodopsin does not change its attachment site, lysine 216, during proton translocation and light-dark adaptation

Abstract

In order to determine the attachment site(s) of the chromophore retinal in bacteriorhodopsin and to examine the possible occurrence of light-induced changes in its binding site, all accessible lysine residues in bacteriorhodopsin were modified by reductive methylation with formaldehyde and NaCNBH3. Upon transformation of the ɛ-amino group of the lysyl residues to the ɛ-N-dimethyl derivatives, these amino acids lose their potential ability to form a Schiff base linkage with retinal. The influence of this modification on the structure and function of bacteriorhodopsin was determined by applying a variety of techniques to modified or native purple membrane sheets which were either in aqueous suspensions, or attached to or incorporated into planar lipid bilayers and lipid vesicles, respectively. No differences were observed between native and methylated bacteriorhodopsin in the kinetics of laser-flash-induced photocurrents and transmission changes, or in the rate and extent of light-induced pH-changes. The overall rate, the activation energy, and the extent of light-dark adaptation were not severely affected by the modification. Furthermore, to identify the attachment site of the chromophore retinal, premethylated bacterioopsin was treated with trinitrobenzenesulphonate. The results show that lysine 216 is the sole attachment site of retinal in bacteriorhodopsin and that a change in this attachment site does not occur during the photocycle, proton translocation or light-dark adaptation. The ɛ-amino groups of the lysine residues do not seem to play a crucial role in the coordinated pathway of proton translocation across bacteriorhodopsin.