ProteinMPNN Recovers Complex Sequence Properties of Transmembrane β-Barrels

Abstract Recent deep-learning (DL) protein design methods have been successfully applied to a range of protein design problems including the de novo design of novel folds, protein binders, and enzymes. However, DL methods have yet to meet the challenge of de novo membrane protein (MP) and the design of complex β-sheet folds. We performed a comprehensive benchmark of one DL protein sequence design method, ProteinMPNN, using transmembrane and water-soluble β-barrel folds as a model, and compared the performance of ProteinMPNN to the new membrane-specific Rosetta Franklin2023 energy function. We tested the effect of input backbone refinement on ProteinMPNN performance and found that given refined and well-defined inputs, ProteinMPNN more accurately captures global sequence properties despite complex folding biophysics. It generates more diverse TMB sequences than Franklin2023 in pore-facing positions. In addition, ProteinMPNN generated TMB sequences that passed state-of-the-art in silico filters for experimental validation, suggesting that the model could be used in de novo design tasks of diverse nanopores for single-molecule sensing and sequencing. Lastly, our results indicate that the low success rate of ProteinMPNN for the design of β-sheet proteins stems from backbone input accuracy rather than software limitations.