MOTIVATION: Protein-protein interactions (PPIs) govern virtually all cellular processes, and a single mutation within a PPI can significantly impact protein functionality, potentially leading to diseases. While numerous approaches have emerged to predict changes in the free energy of binding due to mutations (ΔΔGbind), most lack precision. Recently, protein language models (PLMs) have shown powerful predictive capabilities by leveraging both sequence and structural data from protein complexes, yet they have not been optimized specifically for ΔΔGbind prediction. RESULTS: We developed an approach, ProBASS (Protein Binding Affinity from Structure and Sequence), to predict the effects of mutations on ΔΔGbind using two most advanced PLMs, ESM2 and ESM-IF1, which incorporate sequence and structural features, respectively. We first generated embeddings for each PPI mutant from the two PLMs and then fine-tuned ProBASS by training on a large dataset of experimental ΔΔGbind values. When training and testing were done on the same PPI, ProBASS achieved correlations with experimental ΔΔGbind values of 0.83 ± 0.05 and 0.69 ± 0.04 for single and double mutations, respectively. Additionally, when evaluated on a dataset of 2,325 single mutations across 131 PPIs, ProBASS reached a correlation of 0.81 ± 0.02, substantially outperforming other PLMs in predictive accuracy. Our results demonstrate that refining pre-trained PLMs with extensive ΔΔGbind datasets across multiple PPIs is a successful approach for creating a precise and broadly applicable ΔΔGbind prediction model, facilitating future protein engineering and design studies. ProBASS's accuracy could be further improved through training as more experimental data becomes available. AVAILABILITY AND IMPLEMENTATION: ProBASS is available at: https://colab.research.google.com/github/sagagugit/ProBASS/blob/main/ProBASS.ipynb.

BACKGROUND: Protein-protein interactions (PPIs) are conveyed through binding interfaces or surface patches on proteins that become buried upon binding. Structural and biophysical analysis of many protein-protein interfaces revealed certain unique features of these surfaces that determine the energetics of interactions and play a critical role in protein evolution. One of the significant aspects of binding interfaces is the presence of binding hot spots, where mutations are highly deleterious for binding. Conversely, binding cold spots are positions occupied by suboptimal amino acids and several mutations in such positions could lead to affinity enhancement. While there are many software programs for identification of hot spot positions, there is currently a lack of software for cold spot detection. RESULTS: In this paper, we present Cold Spot SCANNER, a Colab Notebook, which scans a PPI binding interface and identifies cold spots resulting from cavities, unfavorable charge-charge, and unfavorable charge-hydrophobic interactions. The software offers a Py3DMOL-based interface that allows users to visualize cold spots in the context of the protein structure and generates a zip file containing the results for easy download. CONCLUSIONS: Cold spot identification is of great importance to protein engineering studies and provides a useful insight into protein evolution. Cold Spot SCANNER is open to all users without login requirements and can be accessible at: https://colab. RESEARCH: google.com/github/sagagugit/Cold-Spot-Scanner/blob/main/Cold_Spot_Scanner.ipynb .