Insight into how mutations affect protein stability is crucial for protein engineering, understanding genetic diseases, and exploring protein evolution. Numerous computational methods have been developed to predict the impact of amino acid substitutions on protein stability. Nevertheless, comparing these methods poses challenges due to variations in their training data. Moreover, it is observed that they tend to perform better at predicting destabilizing mutations than stabilizing ones. Here, we meticulously compiled a new dataset from three recently published databases: ThermoMutDB, FireProtDB, and ProThermDB. This dataset, which does not overlap with the well-established S2648 dataset, consists of 4038 single-point mutations, including over 1000 stabilizing mutations. We assessed these mutations using 27 computational methods, including the latest ones utilizing mega-scale stability datasets and transfer learning. We excluded entries with overlap or similarity to training datasets to ensure fairness. Pearson correlation coefficients for the tested tools ranged from 0.20 to 0.53 on unseen data, and none of the methods could accurately predict stabilizing mutations, even those performing well in anti-symmetric property analysis. While most methods present consistent trends for predicting destabilizing mutations across various properties such as solvent exposure and secondary conformation, stabilizing mutations do not exhibit a clear pattern. Our study also suggests that solely addressing training dataset bias may not significantly enhance accuracy of predicting stabilizing mutations. These findings emphasize the importance of developing precise predictive methods for stabilizing mutations.
Keywords: computational tools; missense mutations; protein stability changes; stabilizing mutations.
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