Reconstitution of purified halorhodopsin.

AUTOR(ES)

Bogomolni, R A

RESUMO

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 light-induced pH changes followed closely the absorption spectrum. Replacement of chloride by acetate or sulfate causes a shift in the absorption maximum to approximately equal 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.

Documentos Relacionados

• Purification of photochemically active halorhodopsin.
• Isolation and properties of the native chromoprotein halorhodopsin.
• Light-driven proton or chloride pumping by halorhodopsin.
• The photocycle of the chloride pump halorhodopsin. II: Quantum yields and a kinetic model
• The photocycle of the chloride pump halorhodopsin. I: Azidecatalyzed deprotonation of the chromophore is a side reaction of photocycle intermediates inactivating the pump