Effect of Chloride Binding on the Thermal Trimer−Monomer Conversion of Halorhodopsin in the Solubilized System

Abstract

Halorhodopsin from Natronomonas pharaonis (NpHR) acts an inward-directed, light-driven chloride pump and forms a homotrimer. To evaluate effect of trimeric assembly, that is, intermolecular interaction, on the control or modulation of light-driven chloride pumping activity of individual HRs, it is important to understand the thermal and chloride sensitivity of trimer dissociation and the structural stability of HR. In this study, the thermal dissociation of NpHR trimer to monomer in a dodecyl beta-d-maltoside-solubilized system was investigated, using size-exclusion chromatography and visible absorption. In the absence of Cl(-), NpHR retained the trimer assembly at 25 degrees C but dissociated to the monomer with an increase in temperature to >40 degrees C. On the other hand, in the presence of Cl(-), the trimer assembly was maintained at 40 degrees C. The dissociation of the trimer to the monomer after incubation at 40 degrees C, which was determined via size-exclusion chromatography, depended on the Cl(-) concentration and showed a sigmoidal isotherm. From this isotherm, the apparent dissociation constant for Cl(-) was estimated to be 22 mM with a Hill coefficient of 2.2. A similar isotherm was obtained when SO(4)(2-) was used instead of Cl(-) with a dissociation constant of 94 mM. On the other hand, thermal dissociation of the NpHR trimer to the monomer in the absence of Cl(-) proceeded by two components: the fast component is susceptible to the changes in temperature and detergent concentration, and the slow component is accompanied by bleaching at the same time. Activation energies of the fast and slow dissociation components and bleaching were 57.8, 35.3, and 40.5 kcal/mol, respectively. The presence of a second chloride-binding site with a Hill coefficient of approximately 2 at the surface of NpHR to control the trimer-monomer conversion was discussed.

Homotrimer Formation and Dissociation of pharaonis Halorhodopsin in Detergent System

“…Similarly, visual rhodopsin is also dissociated when the protein is solubilized from the rod cell membrane by DDM (36). On the other hand, NpHR trimer functionally expressed on the E. coli membrane is able to retain its trimeric structure even after solubilization by a high concentration of the detergents in the presence of NaCl (8,9,37).…”

“…In BR and HR, CD spectrum in the visible region has been used to help understand the property of retinal chromophore and, in turn, their trimeric assembly (8,9,17,31,32). Recent study has reported that the interaction between the chromophores in the assembly has an exciton coupling and significantly contributes to the CD shape (33).…”

“…On the other hand, it has been recently reported that NpHR overexpressed in Escherichia coli cells, which has no bacterioruberin, has intermolecular interactions robust enough to retain its trimeric assembly upon the solubilization in a detergent DDM solution (8,9). Chloride binding and photocycle of NpHR expressed both in E. coli (17)(18)(19)(20)(21)(22)(23) and in Natronomonas pharaonis (24) cells are the same.…”

“…This oligomerization is a significant process by which information is relayed from outside to inside the cell (5). On the other hand, the ion pumps BR and HR exist as a homotrimer in the archaeal cell membrane (2)(3)(4)(6)(7)(8)(9). This is one of their unique structural properties, but few reports have discussed in detail the mechanisms of trimer formation and stabilization (6)(7)(8)(9).…”

Expression of salinarum halorhodopsin in Escherichia coli cells: Solubilization in the presence of retinal yields the natural state

“…The solubilized NpHR is known to be stable under various conditions, but to be rather easily bleached by continuous illumination under the desalted condition [46]. We now compared the stabilities of the HRs purified from the E. coli membrane.…”