Abstract
The effect of divalent ion binding to deionized bacteriorhodopsin (dI-bR) on the thermal transitions of the protein secondary structure have been studied by using temperature-dependent Fourier transform infrared (FT-IR) spectroscopy. The native metal ions in bR, Ca(2+), and Mg(2+), which we studied previously, are compared with Mn(2+), Hg(2+), and a large, synthesized divalent organic cation, ((Et)(3)N)(2)Bu(2+). It was found that in all cases of ion regeneration, there is a pre-melting, reversible conformational transition in which the amide frequency shifts from 1665 to 1652 cm(-1). This always occurs at approximately 80 degrees C, independent of which cation is used for the regeneration. The irreversible thermal transition (melting), monitored by the appearance of the band at 1623 cm(-1), is found to occur at a lower temperature than that for the native bR but higher than that for acid blue bR in all cases. However, the temperature for this transition is dependent on the identity of the cation. Furthermore, it is shown that the mechanism of melting of the organic cation regenerated bR is different than for the metal cations, suggesting a difference in the type of binding to the protein (either to different sites or different binding to the same site). These results are used to propose specific direct binding mechanisms of the ions to the protein of deionized bR.