A Rationally and Computationally Designed Fluorescent Biosensor for d -Serine

Vanessa Vongsouthi, Jason H. Whitfield, Petr Unichenko, Joshua A. Mitchell, Björn Breithausen, Olga Khersonsky, Leon Kremers, Harald Janovjak, Hiromu Monai, Hajime Hirase, Sarel J. Fleishman, Christian Henneberger, Colin J. Jackson

Research output: Contribution to journalArticlepeer-review

7 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)4193-4205
Number of pages13
JournalACS Sensors
Issue number11
Publication statusPublished - 26 Nov 2021
Externally publishedYes


  • computational design
  • d -serine
  • FRET biosensor
  • neuroimaging
  • protein engineering
  • rational design


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