Fourier Transform Infrared Study of the Effect of Different Cations on Bacteriorhodopsin Protein Thermal Stability

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.

The Role of the Native Lipids and Lattice Structure in Bacteriorhodopsin Protein Conformation and Stability as Studied by Temperature-dependent Fourier Transform-Infrared Spectroscopy

“…The pre-melting transition, however, shows a strong dependence on the lipids whereas it showed no dependence on the pH or the cation with which deionized bR is regenerated (22,23). The origin of this pre-melting transition is still ambiguous at the present time.…”

“…The melting temperature and mechanism were dependent on the identity of the divalent cation indicating that the interaction of the bR with the cation depends on the chemical identity of the cation (23). This is best explained by direct protein-cation binding rather than random surface binding where only the charge density should affect the structure and stability.…”

“…This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. thermal stability was recently studied (22,23). It was shown that, upon cation removal, there is a relatively large change in the protein secondary structure, which is reversed upon regeneration with a number of different divalent cations.…”

“…It was shown that, upon cation removal, there is a relatively large change in the protein secondary structure, which is reversed upon regeneration with a number of different divalent cations. The thermal stability was only partially regenerated, and the extent is dependent on the cation identity (23).…”

“…This effect is also compared with the effect of pH and cations on the protein, which was recently studied (22,23) in an effort to understand the interaction of cations, protein, and lipids.…”