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Genetically encoded fluorescent sugar sensors are valuable tools for the discovery of transporters and for quantitative monitoring of sugar steady-state levels in intact tissues. Genetically encoded Forster resonance energy-transfer sensors for glucose have been designed and optimized extensively, and a full series of affinity mutants is available for in vivo studies. However, to date, only a single improved sucrose sensor FLIPsuc-90μΔ1 with Km for sucrose of ∼90 μM was available. This sucrose sensor was engineered on the basis of an Agrobacterium tumefaciens sugar-binding protein. Here, we took a two-step approach to first improve the dynamic range of the FLIPsuc sensor and then expand the detection range from micro- to millimolar sucrose concentrations by mutating a key residue in the binding site. The resulting series of sucrose sensors may enable investigation of sucrose transporter candidates and comprehensive in vivo analyses of sucrose concentration in plants. Since FLIPsuc-90μ also detects trehalose in animal cells, the new series of sensors will likely be suitable for investigating trehalose transport and monitor trehalose steady-state levels in vivo.

Förster resonance energy transfer (FRET) studies performed at the single molecule level have unique abilities to probe molecular structure, dynamics, and function of biological molecules. This technique requires specimens, like proteins, equipped with two different fluorescent probes attached at specific positions within the molecule of interest. Here, we present an approach of cell-free protein synthesis (CFPS) that provides proteins with two different functional groups for post-translational labeling at the specific amino acid positions. Besides the sulfhydryl group of a cysteine, we make use of an azido group of a p-azido-l-phenylalanine to achieve chemical orthogonality. Herein, we achieve not only a site-specific but, most importantly, also a site-selective, label scheme that permits the highest accuracy of measured data. This is demonstrated in a case study, where we synthesize human calmodulin (CaM) by using a CFPS kit and prove the structural integrity and the full functionality of this protein.

Single-molecule FRET (smFRET) is a powerful tool to investigate conformational changes of biological molecules. In general, smFRET studies require protein samples that are site-specifically double-labeled with a pair of donor and acceptor fluorophores. The common approaches to produce such samples cannot be applied when studying the synthesis and folding of the polypeptide chain on the ribosome. The best strategy is to incorporate two fluorescent amino acids cotranslationally using cell-free protein synthesis systems. Here, we demonstrate the cotranslational site-specific incorporation into a model protein of Atto633, a dye with excellent photophysical properties, suitable for single molecule spectroscopy, together with a second dye using a combination of the sense cysteine and the nonsense amber codon. In this work we show that cotranslational incorporation of good fluorophores into proteins is a viable strategy to produce suitable samples for smFRET studies.

Single-molecule FRET (smFRET) is a powerful tool to investigate molecular structures and conformational changes of biological molecules. The technique requires protein samples that are site-specifically equipped with a pair of donor and acceptor fluorophores. Here, we present a detailed protocol for preparing double-labeled proteins for smFRET studies. The protocol describes two cell-free approaches to achieve a selective label scheme that allows the highest possible accuracy in inter‐dye distance determination.