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1.
ACS Sens ; 6(11): 4193-4205, 2021 11 26.
Article in English | 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 (∼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.


Subject(s)
Biosensing Techniques , Fluorescence Resonance Energy Transfer , Animals , Binding Sites , Ligands , Rats , Serine
2.
ACS Synth Biol ; 9(8): 2107-2118, 2020 08 21.
Article in English | MEDLINE | ID: mdl-32786922

ABSTRACT

Strigolactones are plant hormones and rhizosphere signaling molecules with key roles in plant development, mycorrhizal fungal symbioses, and plant parasitism. Currently, sensitive, specific, and high-throughput methods of detecting strigolactones are limited. Here, we developed genetically encoded fluorescent strigolactone biosensors based on the strigolactone receptors DAD2 from Petunia hybrida, and HTL7 from Striga hermonthica. The biosensors were constructed via domain insertion of circularly permuted GFP. The biosensors exhibited loss of cpGFP fluorescence in vitro upon treatment with the strigolactones 5-deoxystrigol and orobanchol, or the strigolactone analogue rac-GR24, and the ShHTL7 biosensor also responded to a specific antagonist. To overcome biosensor sensitivity to changes in expression level and protein degradation, an additional strigolactone-insensitive fluorophore, LSSmOrange, was included as an internal normalization control. Other plant hormones and karrikins resulted in no fluorescence change, demonstrating that the biosensors report on compounds that specifically bind the SL receptors. The DAD2 biosensor likewise responded to strigolactones in an in vivo protoplast system, and retained strigolactone hydrolysis activity. These biosensors have applications in high-throughput screening for agrochemical compounds, and may also have utility in understanding strigolactone mediated signaling in plants.


Subject(s)
Biosensing Techniques/methods , Heterocyclic Compounds, 3-Ring/analysis , Lactones/analysis , Plant Proteins/metabolism , Biocatalysis , Gene Expression/drug effects , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Heterocyclic Compounds, 3-Ring/metabolism , Heterocyclic Compounds, 3-Ring/pharmacology , Lactones/metabolism , Lactones/pharmacology , Petunia/metabolism , Plant Proteins/antagonists & inhibitors , Plant Proteins/genetics , Protein Domains , Proteolysis/drug effects , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Striga/metabolism
3.
Nat Chem Biol ; 14(9): 861-869, 2018 09.
Article in English | MEDLINE | ID: mdl-30061718

ABSTRACT

Fluorescent sensors are an essential part of the experimental toolbox of the life sciences, where they are used ubiquitously to visualize intra- and extracellular signaling. In the brain, optical neurotransmitter sensors can shed light on temporal and spatial aspects of signal transmission by directly observing, for instance, neurotransmitter release and spread. Here we report the development and application of the first optical sensor for the amino acid glycine, which is both an inhibitory neurotransmitter and a co-agonist of the N-methyl-D-aspartate receptors (NMDARs) involved in synaptic plasticity. Computational design of a glycine-specific binding protein allowed us to produce the optical glycine FRET sensor (GlyFS), which can be used with single and two-photon excitation fluorescence microscopy. We took advantage of this newly developed sensor to test predictions about the uneven spatial distribution of glycine in extracellular space and to demonstrate that extracellular glycine levels are controlled by plasticity-inducing stimuli.


Subject(s)
Fluorescent Dyes/chemistry , Glycine/analysis , Hippocampus/chemistry , Animals , Cells, Cultured , Fluorescence Resonance Energy Transfer , Fluorescent Dyes/chemical synthesis , HEK293 Cells , Humans , Male , Optical Imaging , Rats , Rats, Wistar
4.
Methods Mol Biol ; 1596: 71-87, 2017.
Article in English | MEDLINE | ID: mdl-28293881

ABSTRACT

Small molecule biosensors based on Förster resonance energy transfer (FRET) enable small molecule signaling to be monitored with high spatial and temporal resolution in complex cellular environments. FRET sensors can be constructed by fusing a pair of fluorescent proteins to a suitable recognition domain, such as a member of the solute-binding protein (SBP) superfamily. However, naturally occurring SBPs may be unsuitable for incorporation into FRET sensors due to their low thermostability, which may preclude imaging under physiological conditions, or because the positions of their N- and C-termini may be suboptimal for fusion of fluorescent proteins, which may limit the dynamic range of the resulting sensors. Here, we show how these problems can be overcome using ancestral protein reconstruction and circular permutation. Ancestral protein reconstruction, used as a protein engineering strategy, leverages phylogenetic information to improve the thermostability of proteins, while circular permutation enables the termini of an SBP to be repositioned to maximize the dynamic range of the resulting FRET sensor. We also provide a protocol for cloning the engineered SBPs into FRET sensor constructs using Golden Gate assembly and discuss considerations for in situ characterization of the FRET sensors.


Subject(s)
Luminescent Proteins/chemistry , Luminescent Proteins/genetics , Biosensing Techniques/methods , Carrier Proteins/chemistry , Carrier Proteins/genetics , Fluorescence Resonance Energy Transfer/methods , Phylogeny , Protein Engineering/methods
5.
Protein Sci ; 24(9): 1412-22, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26061224

ABSTRACT

Biosensors for signaling molecules allow the study of physiological processes by bringing together the fields of protein engineering, fluorescence imaging, and cell biology. Construction of genetically encoded biosensors generally relies on the availability of a binding "core" that is both specific and stable, which can then be combined with fluorescent molecules to create a sensor. However, binding proteins with the desired properties are often not available in nature and substantial improvement to sensors can be required, particularly with regard to their durability. Ancestral protein reconstruction is a powerful protein-engineering tool able to generate highly stable and functional proteins. In this work, we sought to establish the utility of ancestral protein reconstruction to biosensor development, beginning with the construction of an l-arginine biosensor. l-arginine, as the immediate precursor to nitric oxide, is an important molecule in many physiological contexts including brain function. Using a combination of ancestral reconstruction and circular permutation, we constructed a Förster resonance energy transfer (FRET) biosensor for l-arginine (cpFLIPR). cpFLIPR displays high sensitivity and specificity, with a Kd of ∼14 µM and a maximal dynamic range of 35%. Importantly, cpFLIPR was highly robust, enabling accurate l-arginine measurement at physiological temperatures. We established that cpFLIPR is compatible with two-photon excitation fluorescence microscopy and report l-arginine concentrations in brain tissue.


Subject(s)
Arginine/chemistry , Biosensing Techniques/methods , Periplasmic Binding Proteins/chemistry , Arginine/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Biosensing Techniques/instrumentation , Computer Simulation , Evolution, Molecular , Fluorescence Resonance Energy Transfer/methods , Fluorescent Dyes/chemistry , Nitric Oxide/metabolism , Optical Imaging/methods , Periplasmic Binding Proteins/genetics , Phylogeny , Protein Engineering/methods , Signal Transduction
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