A computationally designed fluorescent biosensor for D-serine

Periplasmic solute-binding proteins (SBPs) have evolved to balance the demands of ligand affinity, thermostability and conformational change to carry out diverse functions in small molecule transport, sensing and chemotaxis. Although the ligand-induced conformational changes that occur in SBPs make them useful components in biosensors, their complexity can be difficult to emulate and they are challenging targets for protein engineering and design. Here we have engineered a fluorescent biosensor with specificity for the signalling molecule D-serine (D-SerFS) from a D-alanine-specific SBP. Through a combination of binding site and remote mutations, the affinity, specificity and thermostability were optimized to allow the detection of changes in D-serine levels using two-photon excitation fluorescence microscopy in situ and in vivo. This work illustrates the multidimensional constraints that are imposed by the trade-offs between structural dynamics, ligand affinity and thermostability, and how these must be balanced to achieve desirable activities in the engineering of complex, dynamic proteins.