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Generation of circularly permuted fluorescent-protein-based indicators for in vitro and in vivo detection of citrate.

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.

SUBMITTER: Honda Y

PROVIDER: S-EPMC3661591 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Generation of circularly permuted fluorescent-protein-based indicators for in vitro and in vivo detection of citrate.

Honda Yuki Y Kirimura Kohtaro K

PloS one 20130522 5

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. ...[more]

PMID: 23717638

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Project description:Upon blue light irradiation, photoactive yellow protein (PYP) undergoes a conformational change that involves large movements at the N-terminus of the protein. We reasoned that this conformational change might be used to control other protein or peptide sequences if these were introduced as linkers connecting the N- and C-termini of PYP in a circular permutant. For such a design strategy to succeed, the circularly permuted PYP (cPYP) would have to fold normally and undergo a photocycle similar to that of the wild-type protein. We created a test cPYP by connecting the N- and C-termini of wild-type PYP (wtPYP) with a GGSGGSGG linker polypeptide and introducing new N- and C-termini at G115 and S114, respectively. Biophysical analysis indicated that this cPYP adopts a dark-state conformation much like wtPYP and undergoes wtPYP-like photoisomerization driven by blue light. However, thermal recovery of dark-state cPYP is ∼10-fold faster than that of wtPYP, so that very bright light is required to significantly populate the light state. Targeted mutations at M121E (M100 in wtPYP numbering) were found to enhance the light sensitivity substantially by lengthening the lifetime of the light state to ∼10 min. Nuclear magnetic resonance (NMR), circular dichroism, and UV-vis analysis indicated that the M121E-cPYP mutant also adopts a dark-state structure like that of wtPYP, although protonated and deprotonated forms of the chromophore coexist, giving rise to a shoulder near 380 nm in the UV-vis absorption spectrum. Fluorine NMR studies with fluorotryptophan-labeled M121E-cPYP show that blue light drives large changes in conformational dynamics and leads to solvent exposure of Trp7 (Trp119 in wtPYP numbering), consistent with substantial rearrangement of the N-terminal cap structure. M121E-cPYP thus provides a scaffold that may allow a wider range of photoswitchable protein designs via replacement of the linker polypeptide with a target protein or peptide sequence.

Project description:Because the N- and C-terminal amino acids of the catalytic (c) polypeptide chains of Escherichia coli aspartate transcarbamoylase (ATCase) are in close proximity to each other, it has been possible to form in vivo five different active ATCase variants in which the terminal regions of the wild-type c chains are linked in a continuous polypeptide chain and new termini are introduced elsewhere in either of the two structural domains of the c chain. These circularly permuted (cp) chains were produced by constructing tandem pyrB genes, which encode the c chain of ATCase, followed by application of PCR. Chains expressed in this way assemble efficiently in vivo to form active, stable ATCase variants. Three such variants have been purified and shown to have the kinetic and physical properties characteristic of wild-type ATCase composed of two catalytic (C) trimers and three regulatory (R) dimers. The values of Vmax for cpATCase122, cpATCase222, and cpATCase281 ranged from 16-21 mumol carbamoylaspartate per microgram per h, compared with 15 for wild-type ATCase, and the values for K0.5 for the variants were 4-17 mM aspartate, whereas wild-type ATCase exhibited a value of 6 mM. Hill coefficients for the three variants varied from 1.8 to 2.1, compared with 1.4 for the wild-type enzyme. As observed with wild-type ATCase, ATP activated the variants containing the circularly permuted chains, as shown by the lowering of K0.5 for aspartate and a decrease in the Hill coefficient (nH). In contrast, CTP caused both an increase in K0.5 and nH for the variants, just as observed with wild-type ATCase. Thus, the enzyme containing the permuted chains with widely diverse N- and C-termini exhibited the homotropic and heterotropic effects characteristic of wild-type ATCase. The decrease in the sedimentation coefficient of the variants caused by the binding of the bisubstrate ligand N-(phosphonacetyl)-L-aspartate (PALA) was also virtually identical to that obtained with wild-type ATCase, thereby indicating that these altered ATCase molecules undergo the analogous ligand-promoted allosteric transition from the taut (T) state to the relaxed (R) conformation. These ATCase molecules with new N- and C-termini widely dispersed throughout the c chains are valuable models for studying in vivo and in vitro folding of polypeptide chains.

Dataset Information

Generation of circularly permuted fluorescent-protein-based indicators for in vitro and in vivo detection of citrate.

Publications

Generation of circularly permuted fluorescent-protein-based indicators for in vitro and in vivo detection of citrate.

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