Proteins evolve through sequence changes that shape structure, stability, and function. Ancestral sequence reconstruction (ASR) infers ancestral proteins from modern homologs, but many studies focus o..
Proteins evolve through sequence changes that shape structure, stability, and function. Ancestral sequence reconstruction (ASR) infers ancestral proteins from modern homologs, but many studies focus on sequence inference without evaluating structural or energetic feasibility. β-lactamases provide an ideal model due to their evolutionary diversity and clinical relevance in antibiotic resistance. Here, we present an integrated ASR workflow combining phylogenetic inference (IQ-TREE), ancestral reconstruction, and structural and stability validation using AlphaFold and FoldX. By reanalyzing a curated β- lactamase dataset and comparing with a recent study (Risso et al., 2013), we provide a controlled comparison of ancestral sequences, predicted structures, and thermodynamic stability.
