Mayuri Sadoine, Susanne Paradies, Nora Zöllner & Wolf B. Frommer*; Heinrich Heine University Düsseldorf, Mat. Nat Facultät, Institute for Molecular Physiology, Universitätsstr. 1; * Correspondence (no spam): frommew@hhu.de 

The first genetically encoded sugar sensors were developed using Roger Tsien’s concept, in which conformational rearrangements in a calcium binding protein was recorded by FRET changes between two spectral variants of the green fluorescent protein (1) by sandwiching the bacterial periplasmic binding protein (GBP) for maltose or glucose between CFP (cyan) and YFP (yellow)(2, 3). These sensor make use of a ‘clam shell’ movement of the GBP upon glucose binding. However, the dynamic range of these FRET sensors is limited. Roger Tsien’s intensiometric sensors with a much larger dynamic range are based on the use conformation change-induced alteration of Excited State Proton Transfer in circular permutated GFPs (4–6). However, while FRET sensors are ratiometric and thus independent of sensor concentration changes, intensiometric sensors are prone to artifacts, e.g. changes in the amount of sensor due to effects on the promoter driving it. We here therefore developed the Matryoshka concept, in which a reference fluorophore is inserted into the circular permutated GFP, enabling ratiometric analyses that are independent of sensor concentration (5, 6). We here developed a prototype matryoshka-type glucose sensor that has been further improved (Ishikawa & Frommer, in prep.).

Proteins from thermophiles often exhibit enhanced stability. To develop a robust Matryoshka sensor for glucose, the GBP from the thermophile T. thermophilus (ttGBP), for which crystal structures are available, was selected as a scaffold (7, 8). The Looger lab had used ttGBP to generate the intensiometric GluSNFr by inserting cpsfGFP into the T. thermophilus GBP lacking the leader peptide using the linker combination left inker-ProAla/right linker2-AsnPro (L1-PA/L2-NP) between residues 326 and 327 (8). 

Analagous to GluSnFR, the Matryoshka cassette consisting of cpfGFP carrying an insertion of the large Stokes shift LSSmOrange was inserted into a His66A mutant (8) of the mature ttGBP to generate MGlucoMeter1.0 (Fig. 1). 

A construct map in pRSET and the plasmid sequence are shown below (Figure 2 and sequence).

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2. Fehr M, Frommer WB, Lalonde S. 2002. Visualization of maltose uptake in living yeast cells by fluorescent nanosensors. Proc. Natl. Acad. Sci. USA. 99(15):9846–51

3. Fehr M, Lalonde S, Lager I, Wolff MW, Frommer WB. 2003. In vivo imaging of the dynamics of glucose uptake in the cytosol of COS-7 cells by fluorescent nanosensors. J. Biol. Chem. 278(21):19127–33

4. Baird GS, Zacharias DA, Tsien RY. 1999. Circular permutation and receptor insertion within green fluorescent proteins. Proc. Natl. Acad. Sci. U. S. A. 96(20):11241–46

5. Ast C, De Michele R, Kumke MU, Frommer WB. 2015. Single-fluorophore membrane transport activity sensors with dual-emission read-out. eLife. 4:e07113

6. Ast C, Foret J, Oltrogge LM, Michele RD, Kleist TJ, et al. 2017. Ratiometric Matryoshka biosensors from a nested cassette of green- and orange-emitting fluorescent proteins. Nat. Commun. 8(1):431

7. Cuneo MJ, Beese LS, Hellinga HW. 2009. Structural analysis of semi-specific oligosaccharide recognition by a cellulose-binding protein of Thermotoga maritima reveals adaptations for functional diversification of the oligopeptide periplasmic binding protein fold. J. Biol. Chem. 284(48):33217–23

8. Keller JP, Marvin JS, Lacin H, Lemon WC, Shea J, et al. 2021. In vivo glucose imaging in multiple model organisms with an engineered single-wavelength sensor. Cell Rep. 35(12):109284