Prediction of protein biophysical traits from limited data: a case study on nanobody thermostability through NanoMelt

Abstract

1In-silico prediction of protein biophysical traits is often hindered by the limited availability of experimental data and their heterogeneity. Training on limited data can lead to overfitting and poor generalisability to sequences distant from those in the training set. Additionally, inadequate use of scarce and disparate data can introduce biases during evaluation, leading to unreliable model performances being reported. Here, we present a comprehensive study exploring various approaches for protein fitness prediction from limited data, leveraging pre-trained embeddings, repeated stratified nested cross-validation, and ensemble learning to ensure an unbiased assessment of the performances. We applied our framework to introduce NanoMelt, a predictor of nanobody thermostability trained with a dataset of 640 measurements of apparent melting temperature, obtained by integrating data from the literature with 129 new measurements from this study. We find that an ensemble model stacking multiple regression using diverse sequence embeddings achieves state-of-the-art accuracy in predicting nanobody thermostability. We further demonstrate NanoMelts potential to streamline nanobody development by guiding the selection of highly stable nanobodies. We make the curated dataset of nanobody thermostability freely available and NanoMelt accessible as a downloadable software and webserver. 2 Significance StatementRapidly predicting protein biophysical traits with accuracy is a key goal in protein engineering, yet efforts to develop reliable predictors are often hindered by limited and disparate experimental measurements. We introduce a framework to predict biophysical traits using few training data, leveraging diverse machine learning approaches via a semi-supervised framework combined with ensemble learning. We applied this framework to develop NanoMelt, a tool to predict nanobody thermostability trained on a new dataset of apparent melting temperatures. Nanobodies are increasingly important in research and therapeutics due to their ease of production and small size, which allows deeper tissue penetration and seamless combination into multi-specific compounds. NanoMelt outperforms available methods for protein thermostability prediction and can streamline nanobody development by guiding the design and selection of highly stable nanobodies during discovery and optimization campaigns.