CALCIUM AND MAGNESIUM BINDING IN NATIVE AND STRUCTURALLY PERTURBED PURPLE MEMBRANE

The number and identity of the metal cations bound to wild-type bacter iorhodopsin (bR) are determined by using inductively coupled plasma ma ss spectrometry (ICP-MS) and ICP emission techniques. The results indi cate that there at approximate to 2 total Ca2+ and Mg2+ per bR molecul e with a ratio of approximate to 3:1 Ca2+ to Mg2+. This observed ratio is found to agree with the calculated ratio using previously determin ed binding constants for the two high affinity sites of Ca2+ to deioni zed bR (Zhang; et al. Biophys. J. 1992, 61, 1201). This suggests that the high-affinity binding sites in deionized bR are similar to those i n native bR. Structural perturbation of the native membrane by cleavag e of the C-terminus decreases the number of ions per bR to 1.4. The ob served ratio of total ions in this sample to total ions in bR is found to agree with that calculated using known binding constants for each. The results on the number of metal cations/bR and their ratio in bact erioopsin agrees with the calculated number using previously observed binding constants in deionized bO (Yang; et al. Biophys J., in press) only if one assumes that the second high-affinity site (not the first) is removed by retinal removal. Removal of 75% of the lipids from the purple membrane is found to greatly reduce the number of metal cations from 2 to 0.16. This suggest that if metal cations are in the two hig h-affinity sites (which are the only type of binding sites evident in our native bR sample), the removal of lipids, known to change the prot ein tertiary structure, changes also the metal ion binding sites.