Abstract
A one-step purification method for halorhodopsin was developed. Functional proteoliposomes were prepared from this preparation using cholate, which is removed by dialysis in the presence of asolectin or the polar halobacterial lipids. Light-induced outward directed transport of chloride by halorhodopsin was followed by measuring passive proton efflux in the presence of uncoupler; initial rates and extents amounted to significant fractions of values obtained for halorhodopsin-containing cell envelope vesicles. The transport activity was much higher when cholate rather than octyl glucoside was used in the reconstitution. Since CD spectra in cholate but not in octyl glucoside showed band-splitting in the visible region, suggestive of exciton interaction between halorhodopsin monomers, the reconstitution may depend on an aggregate state of the halorhodopsin. The rate constants for three thermal steps in the halorhodopsin photocycle were greatly reduced in the detergent-solubilized samples, but they increased in the proteoliposomes to values similar to those for halorhodopsin in cell envelope vesicles. Thus, the reconstitution yields halorhodopsin with both photochemical and transport activities restored. Freeze-fracture electron micrographs of the proteoliposomes showed unilammellar liposomes with numerous particles of 100-150 A diameter at the fracture faces. These should correspond to halorhodopsin aggregates, formed in the bilayer in an apparently concentration-dependent manner.
Highlights
A one-step purification method for halorhodopsin was developed
Since CD spectra in cholate but not in octyl glucoside showed band-splitting in the visible region, suggestive of exciton interaction between halorhodopsin monomers, the reconstitution may depend on an aggregate state of the halorhodopsin.The rate constants for three thermal steps in the halorhodopsin photocycle were greatly reduced in the detergent-solubilized samples, but they increased in the proteoliposomes to values similar to those for halorhodopsinin cell envelope vesicles
Bacteriorhodopsin forms two-dimensional crystalline arrays in the plane of the cytoplasmic membrane of halobacteria, and work with this protein was greatly helped by the fact that these arrays, or“purple membrane patches,’’can be separated from the rest of the membrane and reconstitutiendto functionally active proteoliposomes (Stoeckenius et al, 1978; Stoeckenius and Bogomolni, 1982)
Summary
Halorhodopsin is one of several retinal proteins found in the cytoplasmic membrane of various halobacteria Some of these pigments have a sensory function, but halorhodopsin functions as alight-driven electrogenic ion pump, to bacteriorhodopsin. The most reproducible methods use polyoxyethylene ether-type detergents, such as Lubrol PX, CIPEB, or Triton X-100 (Steiner and Oesterhelt, 1983; Sugiyama and Mukohata, 1984; Ogurusuet al., 1984).these detergents contain peroxides which cause time-dependent loss of the pigment after purification, and because of the low cmc of these detergents andthe highly hydrophobic nature of halorhodopsin, removal or exchange of these detergents is very difficult. We reporta simple one-step procedure to produce purified halorhodopsin with high recovery, using detergents of high cmc only, and a method for incorporating the halorhodopsin so produced into proteoliposomes with transport activity nearly an order of magnitude higher than obtained before. We describe the properties of the reconstituted halorhodopsin, as regards transport activity, photocycle kinetics, and aggregation state of the protein in the proteoliposomes
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