Directed evolution of Gloeobacter violaceus rhodopsin spectral properties

Area of Science:

• Biophysics
• Molecular Biology
• Protein Engineering

Background:

• Proton-pumping rhodopsins (PPRs) are light-activated proteins crucial for ion transport across membranes.
• PPRs have applications in bioenergy, optogenetics, and as fluorescent sensors, but their spectral properties are not fully understood or engineerable.
• Understanding how protein sequence dictates spectral variation in PPRs is key to their functional engineering.

Purpose of the Study:

• To comprehensively study amino acid substitutions in the retinal-binding pocket of Gloeobacter violaceus rhodopsin (GR).
• To tune the spectral properties of GR and explore the impact of these mutations on proton-pumping activity and fluorescence.
• To identify novel mutations for engineering PPR spectral characteristics.

Main Methods:

• Utilized directed evolution to generate 70 GR variants with altered spectral properties.
• Investigated amino acid substitutions within the retinal-binding pocket of GR.
• Analyzed changes in absorption maxima, proton-pumping activity, and fluorescence of engineered variants.

Main Results:

• Achieved spectral shifts of up to ±80nm in absorption maxima, extending the absorption range beyond natural PPRs.
• Identified single mutations causing 42nm blue shifts and 22nm red shifts without disrupting proton pumping.
• Discovered novel mutations, with 38 not found in known microbial rhodopsins, and identified red-shifted GRs with enhanced fluorescence.

Conclusions:

• Amino acid substitutions in the GR retinal-binding pocket can effectively tune spectral properties.
• Specific mutations can red-shift or blue-shift absorption without compromising essential proton-pumping function.
• Engineered GR variants with novel spectral properties and enhanced fluorescence show promise for future applications.