dF/F0 values for all sensors and controls.

Periplasmic binding proteins (PBPs) are bacterial proteins commonly used as scaffolds for substrate-detecting biosensors. In these biosensors, effector proteins (for example fluorescent proteins) are inserted into a PBP such that the effector protein’s output changes upon PBP-substate binding. The insertion site is often determined by comparison of PBP apo/holo crystal structures, but random insertion libraries have shown that this can miss the best sites. Here, we present a PBP biosensor design method based on residue contact analysis from molecular dynamics. This computational method identifies the best previously known insertion sites in the maltose binding PBP, and suggests further previously unknown sites. We experimentally characterise fluorescent protein insertions at these new sites, finding they too give functional biosensors. Furthermore, our method is sufficiently flexible to both suggest insertion sites compatible with a variety of effector proteins, and be applied to binding proteins beyond PBPs.