A Rationally and Computationally Designed Fluorescent Biosensor for d-Serine.
ACS Sens
; 6(11): 4193-4205, 2021 11 26.
Article
em En
| MEDLINE
| ID: mdl-34783546
ABSTRACT
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 (â¼14%). 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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Base de dados:
MEDLINE
Assunto principal:
Técnicas Biossensoriais
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Transferência Ressonante de Energia de Fluorescência
Idioma:
En
Ano de publicação:
2021
Tipo de documento:
Article