A Structure-guided, Indel-aware Framework for Ancestral Reconstruction of Full-length Seven-transmembrane Proteins

Microbial rhodopsins exhibit diverse functions ranging from ion pumps and ion channels to light sensors, despite sharing a common seven-transmembrane (7TM) domain structure. Their evolutionary process is of interest in understanding how they have acquired such varied functions. A direct route to inferring and experimentally testing plausible ancestral rhodopsins is provided by ancestral sequence reconstruction (ASR). However, ASR of 7TM proteins is often limited in practice 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. The resulting models form high-confidence 7TM folds, including characteristic {beta}-strands and short helices. Finally, both Anc-SzR and Anc-HeR express in Escherichia coli are recovered as stable, colored holoproteins that bind retinal. Together, these results show that full-length, indel-aware ASR can produce experimentally tractable ancestral microbial rhodopsins and enables direct tests of how EM architecture evolves alongside the 7TM core. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/696783v2_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1e46148org.highwire.dtl.DTLVardef@aec0fdorg.highwire.dtl.DTLVardef@1004d7aorg.highwire.dtl.DTLVardef@de9375_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIAncestral schizorhodopsin and heliorhodopsin are reconstructed. C_LIO_LIIndel-aware refining yields compact ancestors with lengths close to extant proteins. C_LIO_LIAlphaFold models retain lineage-specific extra-membrane secondary structures. C_LIO_LIReliability is evaluated by mapping PPs together with AlphaFold confidence. C_LIO_LIAnc-SzR and Anc-HeR express as stable colored holoproteins in E. coli. C_LI