Conversion of a Putative Agrobacterium Sugar-binding Protein into a FRET Sensor with High Selectivity for Sucrose
ABSTRACT Glucose is the main sugar transport form in animals, whereas plants use sucrose to supply non-photosynthetic organs with carbon skeletons and energy. Many aspects of sucrose transport, metabolism, and signaling are not well understood, including the route of sucrose efflux from leaf mesophyll cells and transport across vacuolar membranes. Tools that can detect sucrose with high spatial and temporal resolution in intact organs may help elucidate the players involved. Here, FRET sensors were generated by fusing putative sucrose-binding proteins to green fluorescent protein variants. Plant-associated bacteria such as Rhizobium and Agrobacterium can use sucrose as a nutrient source; sugar-binding proteins were, thus, used as scaffolds for developing sucrose nanosensors. Among a set of putative sucrose-binding protein genes cloned in between eCFP and eYFP and tested for sugar-dependent FRET changes, an Agrobacterium sugar-binding protein bound sucrose with 4 mum affinity. This FLIPsuc-4mu protein also recognized other sugars including maltose, trehalose, and turanose and, with lower efficiency, glucose and palatinose. Homology modeling enabled the prediction of binding pocket mutations to modulate the relative affinity of FLIPsuc-4mu for sucrose, maltose, and glucose. Mutant nanosensors showed up to 50- and 11-fold increases in specificity for sucrose over maltose and glucose, respectively, and the sucrose binding affinity was simultaneously decreased to allow detection in the physiological range. In addition, the signal-to-noise ratio of the sucrose nanosensor was improved by linker engineering. This novel reagent complements FLIPs for glucose, maltose, ribose, glutamate, and phosphate and will be used for analysis of sucrose-derived carbon flux in bacterial, fungal, plant, and animal cells.
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- "No specific sucrose-binding periplasmic binding proteins have been found in bacteria. Since microorganisms in the rhizosphere developed sucrosesensing protein, several candidates from Rhizobium and Agrobacterium were tested, and a homologue of the rhizobial protein (ThuE) from A. tumefacians was found to bind sucrose (Lager et al. 2006). Sucrosebinding protein, ThuE, was then fused to the ECFP and EYFP. "
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; DOI:10.1007/s10529-015-1873-6 · 1.74 Impact Factor
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- "amino acid terminal truncations; aid: ABA interaction domain; cd: catalytic domain; ed, m: enhanced dimerization or monomeric variant DOI: 10.7554/eLife.01741.003 Research article Gu et al., 2006; Lager et al., 2006). Sensory domain proteolysis was circumvented by using a protease-deficient yeast strain (strain BJ5465 lacking Pep4 and Prb1, Figure 2) for expression and purification. "
ABSTRACT: Cytosolic hormone levels must be tightly controlled at the level of influx, efflux, synthesis, degradation and compartmentation. To determine ABA dynamics at the single cell level, FRET sensors (ABACUS) covering a range ∼0.2–800 µM were engineered using structure-guided design and a high-throughput screening platform. When expressed in yeast, ABACUS1 detected concentrative ABA uptake mediated by the AIT1/NRT1.2 transporter. Arabidopsis roots expressing ABACUS1-2µ (Kd∼2 µM) and ABACUS1-80µ (Kd∼80 µM) respond to perfusion with ABA in a concentration-dependent manner. The properties of the observed ABA accumulation in roots appear incompatible with the activity of known ABA transporters (AIT1, ABCG40). ABACUS reveals effects of external ABA on homeostasis, that is, ABA-triggered induction of ABA degradation, modification, or compartmentation. ABACUS can be used to study ABA responses in mutants and quantitatively monitor ABA translocation and regulation, and identify missing components. The sensor screening platform promises to enable rapid fine-tuning of the ABA sensors and engineering of plant and animal hormone sensors to advance our understanding of hormone signaling. DOI: http://dx.doi.org/10.7554/eLife.01741.001eLife Sciences 04/2014; 3:e01741. DOI:10.7554/eLife.01741 · 8.52 Impact Factor
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- "), glucose (Fehr et al., 2003; Deuschle et al., 2006; Takanaga et al., 2008), ribose (Lager et al., 2003), sucrose (Lager et al., 2006), arabinose (Kaper et al., 2008), glutamate (Deuschle et al., 2005; Okumoto et al., 2005; Hires et al., 2008), arginine (Bogner and Ludewig, 2007), glutamine (Yang et al., 2010), histidine, and leucine/isoleucine/valine (Okada et al., 2009) have been developed using PBPs as the ligand-recognition domains. One of the notable insights delivered by the use of metabolite sensors is the amazing heterogeneity of the cellular concentration of metabolites. "
ABSTRACT: Quantitative imaging in live cells is a powerful method for monitoring the dynamics of biomolecules at an excellent spatio-temporal resolution. Such an approach, initially limited to a small number of substrates for which specific dyes were available, has become possible for a large number of biomolecules due to the development of genetically encoded, protein-based sensors. These sensors, which can be introduced into live cells through a transgenic approach, offer the benefits of quantitative imaging, with an extra advantage of non-invasiveness. In the past decade there has been a drastic expansion in the number of biomolecules for which genetically encoded sensors are available, and the functional properties of existing sensors are being improved at a dramatic pace. A number of technical improvements have now made the application of genetically encoded sensors in plants rather straightforward, and some of the sensors such as calcium indicator proteins have become standard analytical tools in many plant laboratories. The use of a handful of probes has already revealed an amazing specificity of cellular biomolecule dynamics in plants, which leads us to believe that there are many more discoveries to be made using genetically encoded sensors. In this short review, we will summarize the progress made in the past 15 years in the development in genetically encoded sensors, and highlight significant discoveries made in plant biology.The Plant Journal 04/2012; 70(1):108-17. DOI:10.1111/j.1365-313X.2012.04910.x · 6.82 Impact Factor