Year: 2020
Type: preprint
Abstract: Summary 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 fluorescent 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 (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.
Institutions: Australian National University, University of Bonn, Weizmann Institute of Science, Australian Regenerative Medicine Institute, Monash University, RIKEN Center for Brain Science, German Center for Neurodegenerative Diseases, UCL Queen Square Institute of Neurology, University College London
Language: English
Subfield: Biophysics
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W3071502199