The effect of metal cation binding on the protein, lipid and retinal isomeric ratio in regenerated bacteriorhodopsin of purple membrane

The effect of metal cation binding on bacteriorhodopsin (bR) in purple memb rane has been examined using in situ attenuated total reflection-Fourier tr ansform infrared difference spectroscopy in aqueous media. It is known that adding metal cations to deionized bR regenerates the purple state from its blue state and recovers the proton pump function, During this process, inf rared spectral changes in the frequency region of 1800-1000 cm(-1) are moni tored. The results reveal that metal cation binding affects the protein con formation, the retinal isomeric composition as well as lipid head groups. I t is also observed that metal cation binding induces conformational changes in the alpha (1)-helix region of bR, converting the portion of its alpha ( 1)-helical domain into beta -turn or disordered coil, In addition, the infl uence of Ho3+ binding on the protein and lipid is observed to be larger tha n that of Ca2+, These results suggest that some of the metal cation binding sites are on the membrane lipid domain, while others could be on the intra helical domain or interhelical loops where the Asp and Glu are located (bin ding with their COO- groups). Our results also suggest that the removal of the C-terminal of bR increase the accessibility of the binding site of meta l cations, which affects protein conformational structure. All these observ ations are discussed in terms of the two proposals given in the literature regarding the metal cation binding sites.