Reconstitution of purified halorhodopsin.

Abstract

Asolectin lipid vesicles containing halorhodopsin show light-induced acidification in the presence of proton ionophores. This effect is abolished by triphenyltin chloride, a chloride/hydroxyl antiporter, and is greatly diminished by valinomycin in the presence of potassium ions, which collapse the membrane potential. This indicates that halorhodopsin orients in the lipid vesicles preferentially inside out, pumping chloride into the extravesicular compartment. The absorption maximum of halorhodopsin in asolectin vesicles in 3 M NaCl is at 567 nm, and the action spectrum for the lightinduced pH changes followed closely the absorption spectrum. Replacement of chloride by acetate or sulfate causes a shift in the absorption maximum to =559 nm and renders the pump inactive. The different photocycles of the two forms were used to show that 80% of the molecules have the extracellular side exposed to the vesicle interior and that the halide-binding site(s) associated with the spectral transition is accessible from the extracellular side of the molecule. The data presented demonstrate that the purified chromoprotein is the light-driven chloride pump in Halobacterium halobium.The membranes of Halobacterium halobium contain at least three retinyl-proteins. Bacteriorhodopsin (bR) and halorhodopsin (hR) are light-driven proton (outward) and chloride (inward) pumps, respectively (1,2). A third pigment, slow rhodopsin (sR), does not mediate light-driven electrogenic ion translocation and it may be the receptor in phototaxis (3). The visible absorption maxima of bR, hR, and sR are at 568 nm, 578 nm, and 587 nm, respectively (4). Both bR and hR undergo cyclic photoreactions with half-times of a few milliseconds (5,6 The photochemical properties of hR are chloride dependent. At physiological chloride concentrations (-3 M), the photocycle half-time is -10 msec and intermediates are observed as maxima in the difference spectrum at 500 nm (P500) and 680 nm (P680) (8).In the light and in the presence of chloride or bromide, hR generates a negative transmembrane electrical potential and protons are driven into the cell. These proton fluxes are greatly enhanced in the presence of proton ionophores and have been used to monitor the chloride transport by hR in intact cells (6, 9) and in cell envelope vesicles (10, 11).In chloride-free medium, the hR absorption maximum shifts to 565 nm (12, 13), and in the photocycle only a redshifted intermediate (P640) has been observed. It shows a maximum of 640 nm in the difference spectrum and decays into the 565 nm ground state with a half-time of -2 msec (14). No light-driven ion transport has been observed under these conditions (2).From the membranes of a bacteriorhodopsin-deficient H. halobium strain, we have recently purified a retinyl-polypeptide of -25 kDa, with the spectroscopic and photochemical properties of hR (15), and similar preparations have been obtained in other laboratories (16,17). However, it remains to be shown that this isolated retinyl-protein is the ion pump and that ...

In Vitro Demonstration of Dual Light-Driven Na+/H+ Pumping by a Microbial Rhodopsin

“…We also demonstrate that Na þ transport by IAR requires Na þ to be present only on the cytoplasmic side of the protein. An inside-out orientation was also found in LUVs of three other microbial rhodopsins, BR (11), HR (12), and PR (13). This orientation has been suggested to be due to the greater number of charged residues at the C-terminal than at the N-terminal region favoring more rapid hydrophobic N-terminal of BR insertion into the preformed LUVs, leading to a more stable state (20).…”

“…Accordingly, we developed an in vitro system for measurement of lightinduced proton fluxes based on sealed large unilamellar vesicles (LUVs) containing purified rhodopsin proteins. LUVs have been previously used to study ion translocation activity in BR (11), halorhodopsin (12), and proteorhodopsin (13).…”

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

“…The reconstitution was performed according to previous reports with some modifications [40,41]. Asolectin (Sigma, St. Louis, MO) was twice purified by acetone/diethyl ether fractionation cycles, and then stored by dissolving in chloroform (100 mg/mL) at -20°C.…”

“…Chloride transport by HsHR was examined as previously described [40,41]. The transport was detected by following the light-induced pH changes that originate from the passive proton movement in response to the membrane potential created by the chloride transport.…”

Photomovement in Motile Microorganisms—ii

“…In this strain another retinal pigment was identified, halorhodopsin (HR), which was then supposed to be the photoreceptor responsible for photophobic reactions (Matsuno-Yagi and Mukohata, 1977;Bogomolni et al, 1984). During its photocycle it forms an intermediate absorbing near 520 nm (Lanyi, 1984).…”