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

Abstract

Salinarum halorhodopsin (HsHR), a light-driven chloride ion pump of haloarchaeon Halobacterium salinarum, was heterologously expressed in Escherichia coli. The expressed HsHR had no color in the E. coli membrane, but turned purple after solubilization in the presence of all-trans retinal. This colored HsHR was purified by Ni-chelate chromatography in a yield of 3-4 mg per liter culture. The purified HsHR showed a distinct chloride pumping activity by incorporation into the liposomes, and showed even in the detergent-solubilized state, its typical behaviors in both the unphotolyzed and photolyzed states. Upon solubilization, HsHR expressed in the E. coli membrane attains the proper folding and a trimeric assembly comparable to those in the native membranes.

Characterization of a Cyanobacterial Chloride-pumping Rhodopsin and Its Conversion into a Proton Pump

“…5A). For other Cl Ϫ pumps, Cl Ϫ binding caused a blue shift (14,34), except for HsHR (22). In contrast, a large red shift occurred for MrHR by its binding of Cl Ϫ , suggesting the differences in the Cl Ϫ binding site from other Cl Ϫ pumps.…”

“…To avoid the contamination of retinal in the photolysed state, the retinal oxime extraction was carried out after 1 min of incubation in the dark. The extraction and the following HPLC analysis was performed as previously described (22).…”

“…Ion Pump Activity Measurements-The MrHR activity was measured in E. coli suspensions using a conventional pH electrode method (22) that detects the pH changes by the pump activity of H ϩ itself or passive H ϩ transfer in response to the membrane potential created by the pump activity for another ion. The E. coli suspensions were prepared as follows.…”

Probing the Cl−-pumping photocycle of pharaonis halorhodopsin: Examinations with bacterioruberin, an intrinsic dye, and membrane potential-induced modulation of the photocycle

“…Furthermore, these vesicles showed high Cl − pumping activity upon light irradiation. This activity was confirmed by the conventional pH electrode method [34]. To impose the K + diffusion potential, the concentrated vesicles were diluted 1:100 into a solution of 3.3 M NaCl containing 50 mM MOPS, pH 7.…”

Existence of two O-like intermediates in the photocycle of <i>Acetabularia</i> rhodopsin II, a light-driven proton pump from a marine alga

“…The 13- cis isomer in the dark-adapted BR has smaller λ max and ɛ values compared with those of the all- trans isomer [21]. Other microbial rhodopsins such as HR from Halobacterium salinarum and Anabaena sensory rhodopsin showed similar characteristic [22,23]. This spectral property is consistent with the assumption that O 1 and O 2 take the RET configuration of 13- cis and all- trans , respectively.…”