Engineering Genetically Encoded Nanosensors for Real-Time In Vivo Measurements of Citrate Concentrations

Cardiff University, United Kingdom
PLoS ONE (Impact Factor: 3.23). 12/2011; 6(12):e28245. DOI: 10.1371/journal.pone.0028245
Source: PubMed


Citrate is an intermediate in catabolic as well as biosynthetic pathways and is an important regulatory molecule in the control of glycolysis and lipid metabolism. Mass spectrometric and NMR based metabolomics allow measuring citrate concentrations, but only with limited spatial and temporal resolution. Methods are so far lacking to monitor citrate levels in real-time in-vivo. Here, we present a series of genetically encoded citrate sensors based on Förster resonance energy transfer (FRET). We screened databases for citrate-binding proteins and tested three candidates in vitro. The citrate binding domain of the Klebsiella pneumoniae histidine sensor kinase CitA, inserted between the FRET pair Venus/CFP, yielded a sensor highly specific for citrate. We optimized the peptide linkers to achieve maximal FRET change upon citrate binding. By modifying residues in the citrate binding pocket, we were able to construct seven sensors with different affinities spanning a concentration range of three orders of magnitude without losing specificity. In a first in vivo application we show that E. coli maintains the capacity to take up glucose or acetate within seconds even after long-term starvation.

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Available from: Sabrina Reich, Oct 10, 2014
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    • "By modifying the residues in the citratebinding pocket, seven mutants were created with different affinities, enhancing the physiological detection range of three orders of magnitude without losing specificity. K d , the ligand concentration at half-maximal saturation, is 8 mM (Ewald et al. 2011). In an in vivo application, E. coli maintains the capacity to take up glucose or acetate within few seconds even after a long-term starvation. "
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    • "We also have adopted the visual analysis method using fluorescent protein in studies of citrate production by A. niger [8], [9], [10]. The major approach to generating these genetically encoded fluorescent indicators is based on fluorescence resonance energy transfer (FRET), and so far many FRET-based indicators for metabolites have been developed (reviewed in [11], e.g., for cAMP [12], ATP [13], glutamate [14], maltose [15], glucose [16], and citrate [17]). On the other hand, some genetically encoded fluorescent indicators employ an alternative approach based on circularly permuted fluorescent proteins (cpFPs), in which the original N and C termini are connected via a peptide linker, and new N and C termini are created in close proximity to the chromophore [18]. "
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