Mass Balance Approximation of Unfolding Improves Potential-Like Methods for Protein Stability Predictions

The prediction of protein stability changes following single-point mutations plays a pivotal role in computational biology, particularly in areas like drug discovery, enzyme reengineering, and genetic disease analysis. Although deep-learning strategies have pushed the field forward, their use in standard workflows remains limited due to resource demands. Conversely, potential-like methods are fast, intuitive, and efficient. Yet, these typically estimate Gibbs free energy shifts without considering the free-energy variations in the unfolded protein state, an omission that may breach mass balance and diminish accuracy. This study shows that incorporating a mass-balance correction (MBC) to account for the unfolded state significantly enhances these methods. While many machine learning models partially model this balance, our analysis suggests that a refined representation of the unfolded state may improve the predictive performance.

JanusDDG: A Thermodynamics-Compliant Model for Sequence-Based Protein Stability via Two-Fronts Multi-Head Attention

Understanding how residue variations affect protein stability is crucial for designing functional proteins and deciphering the molecular mechanisms underlying disease-related mutations. Recent advances in protein language models (PLMs) have revolutionized computational protein analysis, enabling, among other things, more accurate predictions of mutational effects. In this work, we introduce JanusDDG, a deep learning framework that leverages PLM-derived embeddings and a bidirectional cross-attention transformer architecture to predict $\Delta \Delta G$ of single and multiple-residue mutations while simultaneously being constrained to respect fundamental thermodynamic properties, such as antisymmetry and transitivity. Unlike conventional self-attention, JanusDDG computes queries (Q) and values (V) as the difference between wild-type and mutant embeddings, while keys (K) alternate between the two. This cross-interleaved attention mechanism enables the model to capture mutation-induced perturbations while preserving essential contextual information. Experimental results show that JanusDDG achieves state-of-the-art performance in predicting $\Delta \Delta G$ from sequence alone, matching or exceeding the accuracy of structure-based methods for both single and multiple mutations.