Red-shifting mutation of light-driven sodium-pump rhodopsin

Area of Science:

• Microbial rhodopsins
• Optogenetics
• Protein engineering

Background:

• Microbial rhodopsins are light-driven ion transporters crucial for optogenetics.
• Longer-wavelength rhodopsins are needed for reduced phototoxicity and enhanced tissue penetration.
• Controlling neuronal activity with light requires optimized rhodopsin properties.

Purpose of the Study:

• To red-shift the absorption spectrum of a sodium-pump microbial rhodopsin (KR2).
• To investigate the structural and electronic basis for the spectral shift.
• To identify natural microbial rhodopsins with red-shifted absorption.

Main Methods:

• Site-directed mutagenesis of KR2 (P219T/S254A) to alter chromophore environment.
• Fourier transform infrared spectroscopy to analyze structural changes.
• Quantum mechanics/molecular mechanics (QM/MM) modeling to understand electronic properties.

Main Results:

• Achieved a 40-nm red-shift in KR2 absorption without compromising ion-transport activity.
• Observed structural differences in the red-shifted rhodopsin's chromophore.
• QM/MM models revealed altered protein-chromophore electrostatic interactions lowering the excitation energy.

Conclusions:

• Amino acid substitutions can effectively red-shift microbial rhodopsin absorption spectra.
• Understanding protein-chromophore interactions guides the engineering of optogenetic tools.
• A natural red-shifted sodium pump was identified from Jannaschia seosinensis.