Overcoming Protein Orientation Mismatch Enables Efficient Nanoscale Light-Driven ATP Production

Area of Science:

• Synthetic Biology
• Biophysics
• Membrane Protein Engineering

Background:

• Light-driven proton pumps energize adenosine triphosphate (ATP)-producing modules for artificial cells.
• Random orientation of proton pumps limits the efficiency of these artificial systems.
• Uniform protein orientation is crucial for optimizing reconstituted membrane systems.

Purpose of the Study:

• To develop a versatile approach for uniformly orienting light-driven proton pumps in liposomes.
• To enhance the efficiency of ATP production in bottom-up artificial cell assembly.
• To provide a general method for overcoming orientation mismatch in membrane protein reconstitution.

Main Methods:

• Post-translational coupling of proteorhodopsin (pR) to membrane-impermeable domains for guided insertion.
• Development of a novel bifunctional linker (trisNTA-SpyTag) for reversible protein complexation.
• Verification of protein orientation via monitoring vectorial proton pumping and membrane potential generation.

Main Results:

• Achieved uniform orientation of proteorhodopsin (pR) in liposomes.
• Demonstrated highly efficient ATP production when coupled with ATP synthase, driven by inwardly pumping pR.
• Maximal reaction rates were achieved at economical protein concentrations compared to previous modules.

Conclusions:

• The developed technology enables precise control over membrane protein orientation during reconstitution.
• This method significantly enhances the efficiency of light-driven ATP production in artificial cell-like systems.
• The approach is highly customizable, applicable to various membrane proteins, and minimizes genetic modification requirements.