A Rationally and Computationally Designed Fluorescent Biosensor for d-Serine

ACS Sens. 2021 Nov 26;6(11):4193-4205. doi: 10.1021/acssensors.1c01803. Epub 2021 Nov 16.


Solute-binding proteins (SBPs) have evolved to balance the demands of ligand affinity, thermostability, and conformational change to accomplish 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, they are challenging targets for protein engineering and design. Here, we have engineered a d-alanine-specific SBP into a fluorescence biosensor with specificity for the signaling molecule d-serine (D-serFS). This was achieved through binding site and remote mutations that improved affinity (KD = 6.7 ± 0.5 μM), specificity (40-fold increase vs glycine), thermostability (Tm = 79 °C), and dynamic range (∼14%). This sensor allowed measurement of physiologically relevant changes in d-serine concentration using two-photon excitation fluorescence microscopy in rat brain hippocampal slices. This work illustrates the functional trade-offs between protein dynamics, ligand affinity, and thermostability and how these must be balanced to achieve desirable activities in the engineering of complex, dynamic proteins.

Keywords: FRET biosensor; computational design; d-serine; neuroimaging; protein engineering; rational design.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Binding Sites
  • Biosensing Techniques*
  • Fluorescence Resonance Energy Transfer*
  • Ligands
  • Rats
  • Serine


  • Ligands
  • Serine