期刊
ACS SENSORS
卷 6, 期 11, 页码 4193-4205出版社
AMER CHEMICAL SOC
DOI: 10.1021/acssensors.1c01803
关键词
FRET biosensor; D-serine; neuroimaging; protein engineering; rational design; computational design
资金
- Australian Government Research Training Program Scholarship
- ARC
- European Research Council [815379]
- Israel Science Foundation [1844]
- Milner Foundation
- Human Frontiers Science Program (HFSP) [RGY0084/2012]
- German Academic Exchange Service [DAAD-Go8]
- NRW-Ruckkehrerprogramm
- German Research Foundation (DFG) [SFB1089 B03, SPP1757 HE6949/1, FOR2795, HE6949/3, SPP1757]
The study engineered a Dalanine-specific SBP into a fluorescence biosensor for the signaling molecule D-serine, enhancing affinity, specificity, thermostability, and dynamic range through binding site and remote mutations. This sensor allowed measurement of physiologically relevant changes in D-serine concentration.
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 Dalanine-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 mu M), specificity (40-fold increase vs glycine), thermostability (T-m = 79 degrees C), and dynamic range (similar to 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.
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