Conversion of a Putative Agrobacterium Sugar-binding Protein into a FRET Sensor with High Selectivity for Sucrose

Carnegie Institute, Stanford, California, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 11/2006; 281(41):30875-83. DOI: 10.1074/jbc.M605257200
Source: PubMed

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. "
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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. "
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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. "
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