Functional reconstitution of halorhodopsin. Properties of halorhodopsin-containing proteoliposomes.
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.
Duschl
A
A
Department of Physiology and Biophysics, University of California, Irvine 92717.
McCloskey
M A
MA
Lanyi
J K
JK
D000465:Algorithms; D001436:Bacteriorhodopsins / Q000302:isolation & purification*; D002942:Circular Dichroism; D005614:Freeze Fracturing; D006217:Halobacterium / Q000032:analysis; D025602:Halorhodopsins; D008854:Microscopy, Electron; D011510:Proteolipids; D013053:Spectrophotometry
D025602:Halorhodopsins; D011510:Proteolipids; C020485:proteoliposomes; D001436:Bacteriorhodopsins