Conversion of bacteriorhodopsin into a chloride ion pump

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Bacteriorhodopsin is a light-driven proton pump utilizing aspartate-85 for proton transfer.
• Halorhodopsin is a light-driven chloride ion pump, with threonine at the equivalent residue position.
• Understanding residue 85's role is key to elucidating ion transport specificity.

Purpose of the Study:

• To investigate the functional role of residue 85 in bacteriorhodopsin's ion transport mechanism.
• To determine if mutating bacteriorhodopsin's aspartate-85 to threonine alters its ion transport specificity.
• To compare the transport mechanisms of bacteriorhodopsin and halorhodopsin.

Main Methods:

• Site-directed mutagenesis of bacteriorhodopsin to replace aspartate-85 with threonine.
• Expression of the mutated bacteriorhodopsin in Halobacterium salinarium.
• Spectroscopic analysis of the mutated protein's chromophore and photointermediates.
• Functional assays to measure ion transport activity and direction.

Main Results:

• Mutated bacteriorhodopsin with threonine at residue 85 functioned as a chloride ion pump.
• The mutated protein transported chloride ions in the opposite direction of wild-type bacteriorhodopsin.
• Spectroscopic data indicated chloride binding and revealed chloride-dependent photointermediates.
• A common transport mechanism between bacteriorhodopsin and halorhodopsin was supported.

Conclusions:

• The identity of residue 85 critically determines the ionic specificity of these pumps.
• Bacteriorhodopsin and halorhodopsin share a fundamental ion transport mechanism.
• Modifying residue 85 can switch the pump's transported ion from protons to chloride.