Actinorhodopsin: an efficient and robust light-driven proton pump for bionanotechnological applications

Area of Science:

• Microbial rhodopsins
• Protein biochemistry
• Bionanotechnology

Background:

• Actinorhodopsins are microbial rhodopsins (MRs) found in non-marine actinobacteria.
• Their role in energy cycles and potential applications are understudied.
• Understanding actinorhodopsin structure-function is crucial.

Purpose of the Study:

• To characterize the actinorhodopsin RlActR from Rhodoluna lacicola.
• To investigate its properties for potential bionanotechnological uses.
• To determine its oligomeric state and proton pumping efficiency.

Main Methods:

• Overexpression and purification of RlActR in E. coli.
• Spectroscopic analysis and pKa determination.
• Size-exclusion chromatography and cryo-electron microscopy.
• Functional assays using proteoliposomes and E. coli cells.

Main Results:

• RlActR was expressed and purified in milligram quantities with high purity.
• The protein absorbed green light and exhibited a mildly acidic pKa.
• Size-exclusion chromatography indicated a stable oligomeric structure.
• Cryo-EM revealed RlActR forms a pentamer.
• RlActR efficiently established a proton gradient upon light illumination.

Conclusions:

• RlActR is a stable, light-driven proton pump.
• Its pentameric structure is robust.
• RlActR is a promising building block for bionanotechnology.