Computational Design of Periplasmic Binding Protein Biosensors Guided by Molecular Dynamics

Periplasmic binding proteins (PBPs) evolved to scavenge materials from the environment by binding substrate and delivering it to the host bacteria. PBPs are common scaffolds for biosensors: effector proteins, such as circularly permuted green fluorescent protein (cpGFP), can be inserted into a PBP sequence such that the effector protein's output is changed by conformational changes upon PBP substate binding. The site of insertion for maximum output change is often determined by comparison of PBP apo/holo crystal structures, but random insertion libraries have shown that this method can miss the best sites. Here, we present a computational method for identifying insertion sites for cpGFP in the maltose binding PBP that creates functional biosensors. Our method, which uses protein contact analysis, identifies the best-known insertion regions, as well as additional regions that have not been identified previously. We experimentally characterised biosensors with insertions at these regions and found that 75% of designs were functional. This method is generalisable beyond this system and PBPs more generally, and so could be applied to other binding proteins or effector domains. ### Competing Interest Statement The authors have declared no competing interest.