A blue-shifted light-driven proton pump for neural silencing.

Area of Science:

• Optogenetics
• Biophysics
• Molecular Biology

Background:

• Ion-transporting rhodopsins, like Bacteriorhodopsin (BR), are crucial optogenetic tools for controlling neural activity.
• BR absorbs green/red light and acts as a proton pump, inducing hyperpolarization for neural silencing.

Purpose of the Study:

• To engineer a spectrally blue-shifted proton pump for optogenetic neural silencing.
• To investigate the role of conserved residues in spectral tuning and proton pumping activity.

Main Methods:

• Site-directed mutagenesis of conserved residues in a BR homolog (HwBR).
• Spectroscopic analysis to determine absorption maxima (λmax).
• Quantum mechanics/molecular mechanics (QM/MM) calculations to elucidate structural changes.
• Expression of engineered rhodopsins in Caenorhabditis elegans for functional validation.

Main Results:

• Specific mutations in HwBR caused a significant blue shift in absorption (λmax = 498 nm) while maintaining proton pumping activity.
• QM/MM calculations revealed chromophore rotation and disrupted π conjugation in the mutant.
• Similar spectral blue shifts were achieved in archearhodopsin-3 (Arch).
• Engineered Arch variants effectively silenced neural activity in C. elegans upon blue light illumination.

Conclusions:

• Conserved residues around the retinal chromophore are critical for spectral tuning in BR homologs.
• Engineered blue-shifted proton pumps offer new possibilities for optogenetic applications.
• The developed blue-shifted rhodopsin is a viable tool for neural silencing using blue light in vivo.