Alchemical Free-Energy Framework for Multiresidue Reoptimization of Antimicrobial Peptide-Membrane Affinity.
Antimicrobial peptide (AMP) design is commonly guided by sequence-level descriptors such as cationicity, hydrophobicity, and amphipathicity. However, this design paradigm often relies on empirical intervention or machine-learning prediction, lacks direct support from rigorous quantitative thermodynamic data, and remains difficult to apply to the optimization of known AMP sequences. Here, using the recently proposed WTM-λABF method, we develop an alchemical free-energy framework that incorporates peptide-membrane affinity calculations into sequence reoptimization. Owing to the enhanced sampling efficiency of WTM-λABF, this framework can efficiently handle densely discretized alchemical pathways required for cooperative multiresidue mutations, thereby enabling simultaneous multiresidue substitutions to be evaluated within a single alchemical transformation in each environment, without decomposition into separate single-residue transformations. Following the empirical proposal of candidate mutations, mutation-induced ΔΔG values are used as a membrane-affinity thermodynamic criterion for their interpretation, classification, and prioritization prior to experimental validation. We first validate the framework using a literature-reported membrane-active peptide series, where the calculated ΔΔG values are interpreted together with experimental activity trends and physicochemical descriptors. We then apply the framework to the iterative reoptimization of AMP sequences identified in our previous work. Stepwise multiresidue mutations are first proposed based on empirical design principles and subsequently evaluated using WTM-λABF, thereby identifying mutations that provide favorable contributions to relative membrane affinity and yielding new candidate AMP sequences for subsequent experimental screening. By extending the previously developed WTM-λABF method to mutation-dependent membrane-insertion thermodynamics in medium-length AMP-membrane systems, this work establishes a physically grounded framework that integrates empirical AMP redesign with free-energy-based thermodynamic evaluation of multiresidue reoptimization.