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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    ABSTRACT: Neighboring cells in the same tissue can exist in different states of dynamic activities. After genomics, proteomics and metabolomics, fluxomics is now equally important for generating accurate quantitative information on the cellular and sub-cellular dynamics of ions and metabolite, which is critical for functional understanding of organisms. Various spectrometry techniques are used for monitoring ions and metabolites, although their temporal and spatial resolutions are limited. Discovery of the fluorescent proteins and their variants has revolutionized cell biology. Therefore, novel tools and methods targeting sub-cellular compartments need to be deployed in specific cells and targeted to sub-cellular compartments in order to quantify the target-molecule dynamics directly. We require tools that can measure cellular activities and protein dynamics with sub-cellular resolution. Biosensors based on fluorescence resonance energy transfer (FRET) are genetically encoded and hence can specifically target sub-cellular organelles by fusion to proteins or targetted sequences. Since last decade, FRET-based genetically encoded sensors for molecules involved in energy production, reactive oxygen species and secondary messengers have helped to unravel key aspects of cellular physiology. This review, describing the design and principles of sensors, presents a database of sensors for different analytes/processes, and illustrate examples of application in quantitative live cell imaging.
    Biotechnology Letters 07/2015; 37(10). DOI:10.1007/s10529-015-1873-6 · 1.59 Impact Factor
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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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    ABSTRACT: Indicators for citrate, particularly those applicable to its in vivo detection and quantitation, have attracted much interest in both biochemical studies and industrial applications since citrate is a key metabolic intermediate playing important roles in living cells. We generated novel fluorescence indicators for citrate by fusing the circularly permuted fluorescent protein (cpFP) and the periplasmic domain of the bacterial histidine kinase CitA, which can bind to citrate with high specificity. The ratiometric fluorescent signal change was observed with one of these cpFP-based indicators, named CF98: upon addition of citrate, the excitation peak at 504 nm increased proportionally to the decrease in the peak at 413 nm, suitable for build-in quantitative estimation of the binding compound. We confirmed that CF98 can be used for detecting citrate in vitro at millimolar levels in the range of 0.1 to 50 mM with high selectivity; even in the presence of other organic acids such as isocitrate and malate, the fluorescence intensity of CF98 remains unaffected. We finally demonstrated the in vivo applicability of CF98 to estimation of the intracellular citrate concentration in Escherichia coli co-expressing the genes encoding CF98 and the citrate carrier CitT. The novel indicator CF98 can be a specific and simple detection tool for citrate in vitro and a non-invasive tool for real-time estimation of intracellular concentrations of the compound in vivo.
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