EvoMut: A Computational Framework for Engineering Oxidative Stability in Proteins

Abstract Amino acid oxidation is a major cause of protein instability and loss of function in therapeutic and industrial settings. Although methionine, cysteine, tyrosine, and tryptophan residues are widely recognized as oxidation-prone, only a subset of such residues are dominant functional hotspots, and not all are suitable targets for mutation. Identifying these vulnerable yet engineerable sites remains a major challenge. Here, we present EvoMut, a residue-level analytical framework for evaluating both oxidative vulnerability and mutation feasibility. EvoMut estimates oxidation risk by integrating structural features, local functional context, intrinsic chemical susceptibility, and evolutionary conservation. A central feature of the framework is the explicit separation of oxidation risk from mutation feasibility: candidate substitutions are evaluated only after high-risk residues are identified and ranked by evolutionary substitution patterns. Application of EvoMut to multiple proteins, and evaluation with experimental data, showed that oxidation-prone residues differ markedly in their engineering potential. EvoMut distinguishes residues that are both oxidation- sensitive and evolutionarily permissive from those that are chemically vulnerable but functionally constrained. By providing residue-level mechanistic insight, EvoMut offers a practical framework for the rational design of oxidation-resistant proteins. EvoMut is freely available as a web server at https://evomut.org. Significance Statement Strategies to improve oxidative stability in proteins often rely on chemical intuition or solvent accessibility alone, with limited consideration of functional and evolutionary constraints. EvoMut addresses this gap by explicitly separating oxidative vulnerability from mutation feasibility and integrating structural, chemical, functional, and evolutionary information within an interpretable framework. It helps explain why some oxidation-prone residues can be successfully engineered whereas others remain constrained; thus, supporting rational decision-making in oxidative stability engineering. Competing Interest Statement The authors have declared no competing interest.