Poster Presentation The 44th Lorne Conference on Protein Structure and Function 2019

Engineering a FRET-based biosensor for visualising hippocampal D-serine (#202)

Vanessa Vongsouthi 1 , Jason Whitfield 2 , Christian Henneberger 3 , Colin Jackson 1
  1. Australian National University, Canberra, ACT, Australia
  2. Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD, Australia
  3. Institute of Cellular Neurosciences, University of Bonn, Bonn, Germany

The action of D-serine as a transmitter molecule in the nervous system has attracted considerable attention in recent years. As a co-agonist required for the activation of N-methyl-D-aspartate receptors (NMDARs), the molecule is thought to play a key role in the mechanism of synaptic plasticity1. However, our ability to study D-serine dynamically in brain tissue has been limited by the lack of an optical biosensor that is non-invasive and specific, such as the Förster Resonance Energy Transfer (FRET)-based biosensor for glycine2. The central issue that has prevented the construction of a similar FRET-based sensor for D-serine is the absence of any naturally occurring, and specific, D-serine binding proteins. To overcome this, we have employed computational protein design to engineer an existing D-alanine/glycine binding protein (DalS)3 towards increased D-serine specificity. Fusion of this engineered binding domain to a pair of donor/acceptor fluorescent proteins created the first genetically encodable FRET-based biosensor for D-serine. Several iterations of computational design and experimental characterisation produced a sensor with high thermostability (Tm = 79 °C) and affinity for D-serine (Kd = 7 μM). This sensor was successfully applied in rat brain slices to visualise changes in hippocampal D-serine levels using two-photon excitation (2PE) fluorescence microscopy. It is hoped that it will become a widely used experimental tool that could yield new insight into the dynamic role of D-serine in synaptic plasticity, learning and memory formation.

  1. Van Horn, M. R., Sild, M. & Ruthazer, E. S. D-serine as a gliotransmitter and its roles in brain development and disease. Front. Cell. Neurosci. 7, 39 (2013).
  2. Zhang, W. H. et al. Monitoring hippocampal glycine with the computationally designed optical sensor GlyFS. Nat. Chem. Biol. 14, 861–869 (2018).
  3. Osborne, S. E. et al. Characterization of DalS, an ATP-binding cassette transporter for D-alanine, and its role in pathogenesis in Salmonella enterica. J. Biol. Chem. 287, 15242–15250 (2012).