The Effects of Salt on the Stability of the Light-driven Chloride Pump, halorhodopsin
Yasuo SUGIYAMA*
Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-01, Japan
Extremely halophilic archaebacteria grow in a medium containing 25% (w/v) NaC1 and balance the external high salt concentration by accumulating within the cell the inorganic ions such as KC1. Therefore, all the cellular components have to be adapted to function at the extremely high intracellular concentration of salt. X-ray crystallography of halophilic malate dehydrogenase and ferredoxin revealed the structural features that promote the stability of the soluble proteins at the high salt concentrations. On the other hand, little is known about the stability of the membrane-embedded proteins under the high salt conditions. To address this issue, we compared the stability of halorhodopsin/purple membrane (hR/PM) with that of bacteriorhodopsin/purple membrane (bR/PM). In this work, we studied the effects of chaotropic ions and SDS on bR/PM and hR/PM. Furthermore, the thermal stability of hR/PM and the purified hR was examined to find out a condition of crystallization.
1. Low concentrations of SDS (lower than) induced the blue form of bR/PM in a reversible manner. When the concentrations of SDS increased to higher than cmc, bR/PM was denatured via an intermediate with λmax = 450 nm. In contrast to bR/PM, the isothermal chromophore bleaching (denaturation) of hR/PM was accelerated by low concentrations of SDS because the 570 nm chromophore directly changed to the 380 nm species (free retinal) without an intermediate. This indicated that the binding of SDS monomer changed the tertiary structure of hR/PM, which led to the hydrolysis of the retinal Schiff base linkage.
2. Whereas bR/PM was stable at 80℃, hR/PM irreversibly denatured even at 4℃ and the denaturation rate increased with increasing the temperature. The denaturation proceeded with two independent processes (fast and slow phases) and the fraction of fast phase increased at higher temperature. Increasing the concentrations of salt or decreasing the concentrations of MEGA-9 diminished the isothermal denaturation of purified hR. Then the micelle structure of the detergents and/or lipid would affect the stability of hR, and salt may restrict the thermal motion of these mixed micelle structure.
