# Elucidation of Expression Patterns and Functional Properties of Archaerhodopsin Derived from Halorubrum sp. Ejinoor

## TL;DR

This study introduces HeAR, a new optogenetic tool from an archaeon, which offers improved light-driven proton pumping with reduced phototoxicity and better control for neuroscience.

## Contribution

HeAR provides a red-shifted, bistable proton pump with minimal phototoxicity, enabling multi-channel neural control and overcoming current optogenetic limitations.

## Key findings

- HeAR exhibits a red-shifted absorption peak at 550 nm and prolonged dark-adaptation of 160 minutes.
- AlphaFold modeling shows HeAR has an optimized retinal-binding architecture with high proton-pumping efficiency.
- HeAR’s spectral specificity avoids overlap with excitatory opsins, enabling non-invasive multi-channel neuromodulation.

## Abstract

This study introduces Archaerhodopsin (HeAR), a light-driven proton pump from the salt-tolerant archaeon Halorubrum sp. Ejinoor, as a next-generation optogenetic tool. HeAR shares structural similarities with bacteriorhodopsin (BR), forming stable trimers, but exhibits a red-shifted absorption peak (550 nm) and prolonged dark-adaptation (160 min), enabling sustained neuronal inhibition with minimal phototoxicity. Through AlphaFold modeling and biophysical assays, we demonstrate HeAR’s optimized retinal-binding architecture, which enhances proton-pumping efficiency and stability under physiological conditions. Unlike existing tools, HeAR’s activation window (550–560 nm) avoids spectral overlap with excitatory opsins, permitting multi-channel neural control. Discovered in Inner Mongolian salt lakes, HeAR bridges extremophile adaptations and optogenetic engineering, offering a robust platform for non-invasive neuromodulation and therapeutic development. Its unique properties address key limitations in current optogenetics, such as photodamage and temporal precision, positioning HeAR as a transformative tool for neuroscience research.

This study elucidates the structural determinants and optogenetic potential of Archaerhodopsin HeAR, a proton pump from Halorubrum sp. Ejinoor isolated from Inner Mongolian salt lakes. Through heterologous expression in E. coli BL21 (DE3) and integrative biophysical analyses, we demonstrate that HeAR adopts a stable trimeric architecture (129 kDa) with detergent-binding characteristics mirroring bacteriorhodopsin (BR); however, it exhibits a 10 nm bathochromic spectral shift (λmax = 550 nm) and elevated proton affinity (Asp-95 pKa = 3.5 vs. BR Asp-85 pKa = 2.6), indicative of evolutionary optimization in its retinal-binding electrostatic microenvironment. Kinetic profiling reveals HeAR’s prolonged photocycle (100 ms vs. BR’s 11 ms), marked by rapid M-state decay (3.3 ms) and extended dark-adaptation half-life (160 min), a bistable behavior attributed to enhanced hydrogen bond persistence (80%) and reduced conformational entropy (RMSD = 2.0 Å). Functional assays confirm light-driven proton extrusion (0.1 ng H⁺/mg·s) with DCCD-amplified flux (0.3 ng H⁺/mg·s) and ATP synthesis (0.3 nmol/mg·s), underscoring its synergy with H⁺-ATPase. Phylogenetic and structural analyses reveal 95% homology with Halorubrum AR4 and conservation of 11 proton-wire residues, despite divergent Trp/Tyr/Ser networks that redefine chromophore stabilization. AlphaFold-predicted models (TM-score > 0.92) and molecular docking identify superior retinoid-binding affinity (ΔG = −12.27 kcal/mol), while spectral specificity (550–560 nm) and acid-stable photoresponse highlight its adaptability for low-irradiance neuromodulation. These findings position HeAR as a precision optogenetic tool, circumventing spectral overlap with excitatory opsins and enabling sustained hyperpolarization with minimized phototoxicity. By bridging microbial energetics and optobioengineering, this work expands the archaeal rhodopsin toolkit and provides a blueprint for designing wavelength-optimized photoregulatory systems.