Resurrecting Full-length Ancestral Schizorhodopsins and Heliorhodopsins with Structure-guided, Indel-aware Sequence Reconstruction

Abstract Microbial rhodopsins exhibit diverse functions ranging from ion pumps and channels to light sensors, despite sharing a common seven-transmembrane (7TM) architecture. Understanding how this functional diversity evolved is a long-standing problem, and ancestral sequence reconstruction (ASR) offers a direct route to inferring and experimentally testing plausible ancestral rhodopsins. However, ASR of 7TM proteins is often limited by alignment ambiguity and insertion–deletion (indel) uncertainty, especially in extra-membrane (EM) loops and termini. As a result, many studies focus on trimmed transmembrane (TM) cores and treat EM regions by manual curation, leaving the evolutionary history of full-length architecture difficult to test experimentally. Here we reconstruct and resurrect full-length ancestral schizorhodopsins (Anc-SzR) and heliorhodopsins (Anc-HeR), two microbial rhodopsin families that share a retinal-binding 7TM core but differ in membrane topology and EM secondary-structure elements. Starting from untrimmed alignments, we combine structure-consistent multiple sequence alignments and profile-based evolutionary models with an explicit indel-aware refinement that merges amino-acid ancestral states with binary ancestral gap inference on a fixed topology. Indel-aware refinement prevents artificially overextended ancestors and yields compact full-length sequences. AlphaFold3 predictions for the indel-corrected ancestors support high-confidence 7TM folds and recover lineage-specific EM features, including characteristic β-strands and short helices. Both Anc-SzR and Anc-HeR can be ex-pressed in Escherichia coli and recovered as stable, colored, retinal-binding holoproteins. In a whole-cell pH assay, Anc-SzR shows light-driven proton-transport activity, whereas Anc-HeR shows no detectable ion-pumping signal, consistent with extant heliorhodopsins. Together, these results show that full-length, indel-aware ASR can produce experimentally tractable ancestral microbial rhodopsins and enable direct tests of how EM architecture evolves alongside the 7TM core. Competing Interest Statement The authors have declared no competing interest. Footnotes - Added ESR proxy validation (SzR4, HeR 48C12; TM/EM-resolved) - Added gappyout baseline comparison - Added whole-cell pH assay (with or without CCCP) for Anc-SzR / Anc-HeR