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

This study elucidates the structural determinants and optogenetic potential of Archaerhodopsin <i>He</i>AR, a proton pump from <i>Halorubrum</i> sp. Ejinoor isolated from Inner Mongolian salt lakes. Through heterologous expression in <i>E. coli</i> BL21 (DE3) and integrative biophysical analyses, we demonstrate that <i>He</i>AR 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 <i>He</i>AR'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 <i>Halorubrum</i> 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 <i>He</i>AR 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.