The C-terminus and the Ca2+ low-affinity binding sites in bacteriorhodopsin

Abstract

Bacteriorhodopsin (bR) is found previously to have two high-affinity and four to six low-affinity Ca2+ binding sites. Our previous studies with site-directed mutation suggested that the two high-affinity sites are located within the protein and close to the retinal pocket. In order to investigate the location of the four to six low-affinity binding sites, we studied Ca2+ binding to deionized bR as well as its variant in which its C-terminus is removed by papain treatment. Potentiometric titration with Ca(2+)-selective electrodes were carried out and Scatchard plots were obtained from the titration data. We found that most of the low-affinity sites are eliminated upon removal of the C-terminus in bR. This suggests that the low-affinity sites in bR are on the surface. The involvement of the C-terminus in these sites is discussed.