A Rationally and Computationally Designed Fluorescent Biosensor for d‑Serine

• Published in: ACS sensors 2021-11, Vol.6 (11), p.4193-4205
• Main Authors: Vongsouthi, Vanessa, Whitfield, Jason H, Unichenko, Petr, Mitchell, Joshua A, Breithausen, Björn, Khersonsky, Olga, Kremers, Leon, Janovjak, Harald, Monai, Hiromu, Hirase, Hajime, Fleishman, Sarel J, Henneberger, Christian, Jackson, Colin J
• Format: Article
• Language: English
• Subjects: Animals; Binding Sites; Biosensing Techniques; Fluorescence Resonance Energy Transfer; Ligands; Rats; Serine
• ISSN: 2379-3694; 2379-3694
• DOI: 10.1021/acssensors.1c01803

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 (K D = 6.7 ± 0.5 μM), specificity (40-fold increase vs glycine), thermostability (T m = 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.