Machine learning models for predicting protein condensate formation from sequence determinants and embeddings

Intracellular phase separation of proteins into biomolecular condensates is increasingly recognised as an important phenomenon for cellular compartmentalisation and regulation of biological function. Different hypotheses about the parameters that determine the tendency of proteins to form condensates have been proposed with some of them probed experimentally through the use of constructs generated by sequence alterations. To broaden the scope of these observations, here, we established an in silico strategy for understanding on a global level the associations between protein sequence and condensate formation, and used this information to construct machine learning classifiers for predicting liquid–liquid phase separation (LLPS) from protein sequence. Our analysis highlighted that LLPS–prone sequences are more disordered, hydrophobic and of lower Shannon entropy than sequences in the Protein Data Bank or the Swiss-Prot database, and have their disordered regions enriched in polar, aromatic and charged residues. Using these determining features together with neural network based word2vec sequence embeddings, we developed machine learning classifiers for predicting protein condensate formation. Our model, trained to distinguish LLPS-prone sequences from structured proteins, achieved high accuracy (93%; 25-fold cross-validation) and identified condensate forming sequences from external independent test data at 97% sensitivity. Moreover, in combination with a classifier that had developed a nuanced insight into the features governing protein phase behaviour by learning to distinguish between sequences of varying LLPS propensity, the sensitivity was supplemented with high specificity (approximated ROC–AUC of 0.85). These results provide a platform rooted in molecular principles for understanding protein phase behaviour. The predictor is accessible from https://deephase.ch.cam.ac.uk/. Significance Statement The tendency of many cellular proteins to form protein-rich biomolecular condensates underlies the formation of subcellular compartments and has been linked to various physiological functions. Understanding the molecular basis of this fundamental process and predicting protein phase behaviour have therefore become important objectives. To develop a global understanding of how protein sequence determines its phase behaviour, here, we constructed bespoke datasets of proteins of varying phase separation propensity and identified explicit biophysical and sequence-specific features common to phase separating proteins. Moreover, by combining this insight with neural network based sequence embeddings, we trained machine learning classifiers that identified phase separating sequences with high accuracy, including from independent external test data. The predictor is available from https://deephase.ch.cam.ac.uk/.