The trehalose/maltose-binding protein as the sensitive element of a glucose biosensor A.V. Fonin a,⇑ , O.I. Povarova a , M. Staiano b , S. D’Auria b , K.K. Turoverov a,c , I.M. Kuznetsova a,c a Institute of Cytology of the Russian Academy of Sciences, St. Petersburg, Russia b CNR, Laboratory for Molecular Sensing, IBP, 111 80131 Naples, Italy c St. Petersburg State Polytechnical University, St. Petersburg, Russia article info Article history: Available online 23 January 2014 Keywords: Fluorescence Biosensor Glucose Fluorescent dye Stability abstract The promising direction of the development of a modern glucometer is the construction of sensing element on the basis of stained (dyed) protein which changes its fluorescence upon glucose binding. One of the proteins that can be used for this purpose is the D-trehalose/D-maltose-binding protein (TMBP) from the thermophilic bacteria Thermococcus litoralis. We investigated the physical–chemical properties of the protein and evaluated its stability to the denaturing action of GdnHCl and heating. It was confirmed that TMBP is an extremely stable protein. In vivo, the intrinsic ligands of TMBP are trehalose and maltose, but TMBP can also bind glucose. The dissociation constant of the TMBP–glucose complex is in the range of 3–8 mM. The binding of glucose does not noticeably change the intrinsic fluorescence of the TMBP. To register protein-glucose binding, we used the fluorescence of the thiol-reactive dye BADAN attached to TMBP. Because the fluorescence of BADAN attached to the cysteine Cys182 of TMBP does not change upon glucose binding, the mutant forms TMBP/C182S/X_Cys were created. In these mutant proteins, Cys182 is replaced by Ser, removing intrinsic binding site of BADAN and a new dye binding sites were introduced. The largest increase (by 1.4 times) in the intensity of the dye fluorescence was observed upon TMBP/ C182S/A14C-BADAN–Glc complex formation. The dissociation constant of this complex is 3.4 ± 0.1 mM. We consider TMBP/C182S/A14C mutant form with attached fluorescent dye BADAN as a good basis for further research aimed to develop of series of TMBP mutant forms with different affinities to glucose labeled with fluorescent dyes. Ó 2014 Elsevier B.V. All rights reserved. 1. Introduction In recent decades, the number of people with diabetes has been continuously increasing. A continuous or a sufficiently regular monitoring of sugar level in the blood of diabetic patients is needed to prevent the negative effects of hyper- and hypoglycemia. A promising direction for the creation of a non-invasive glucometer is the development of a biosensor system with a protein sensitive element those interaction with glucose could be reversible. Exam- ples of such proteins are lectins and periplasmic ligand-binding proteins (PBPs) that interact with glucose [1]. The tertiary struc- ture of PBPs is significantly changed upon the interaction with glucose. One of the most widely used methods for the detection of proteins’ conformational changes is fluorescence. D-glucose/ D-galactose-binding protein (GGBP) from E.coli belonging to the PBP class is commonly used for the development of the glucose- sensing element. This protein has a high affinity for glucose (K d = 1 lM), and upon interaction with glucose, its spatial structure undergoes significant changes. Several mutant forms of GGBP with various extrinsic probes that fluoresce in the visible [2,3] and in the infrared region of the spectrum [4,5], as well as a number of FRET- constructions [6,7], were created. At the same time, investigations in this direction face several problems, such as a relatively low sta- bility of GGBP [8,9] and low value of glucose dissociation constant. Both problems can be solved using another PBP–TMBP from hyper- thermophilic bacteria Thermococcus litoralis. Intrinsic ligands of this protein are trehalose and maltose, but TMBP can also bind glu- cose with a significantly greater dissociation constant of the pro- tein-glucose complex (3 – 8 mM) compared to that of GGBP [10]. The aim of this work was to examine the possibility of using TMBP as the sensitive element of a glucose biosensor system. 2. Experimental 2.1. Materials D-glucose (Sigma, USA), guanidine hydrochloride (GdnHCl; Nacalai Tesque, Japan) and a fluorescent dye BADAN (AnaSpec, USA) were used without additional purification. To determine the 0925-3467/$ - see front matter Ó 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.optmat.2014.01.001 ⇑ Corresponding author. Tel.: +7 8122971957. E-mail address: alexfonin@incras.ru (A.V. Fonin). Optical Materials 36 (2014) 1676–1679 Contents lists available at ScienceDirect Optical Materials journal homepage: www.elsevier.com/locate/optmat