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The conformational changes of the Escherichia coli glutamine-binding protein (GlnBP) induced by GdnBP and the effect of the binding of glutamine (Gln) on these processes is studied. The use of the parametric presentation of fluorescence data showed that both GlnBP unfolding and GlnBP-Gn unfolding are three-step processes (N->I(sub 1)->I9sub 2)->U), though in the case of the GlnBP-Gln complex these stages essentially overlap.

Millions of tons of pesticides are applied worldwide annually in agriculture. Among them, herbicides such as triazines and ureas, originating from agricultural runoff, can contaminate soils and surface and ground waters with severe toxic effects on humans. Nowadays, different analytical techniques are available for the detection of these chemicals; however, most of them are expensive and time-consuming, especially in the case of routine analyses. For this reason, on the basis of results collected through many years of experience in the field of photosynthetic organisms, we designed a biosensor platform intended for the easy, low-cost, and fast prescreening of photosynthetic herbicides. The platform combines the possibilities of amperometric and optical transduction systems, which have proven to be highly sensitive (limits of detection = 10(-10)-10(-8) M). The use of genetically modified algae strengthens the power of the platform, allowing different subclasses of herbicides to be recognized. The system has been validated for the analysis of environmental water and is proposed to support laboratories involved in the control of water pollution.

The D-glucose/D-galactose-binding protein (GGBP) of Escherichia coli serves as an initial component for both chemotaxis toward D-galactose and D-glucose and high-affinity active transport of the two sugars. GGBP is a monomer with a molecular weight of about 32 kDa that binds glucose with micromolar affinity. The sugar-binding site is located in the cleft between the two lobes of the bilobate protein. In this work, the local and global structural features of GGBP were investigated by a strategic fluorescence labeling procedure and spectroscopic methodologies. A mutant form of GGBP containing the amino acid substitution Met to Cys at position 182 was realized and fluorescently labeled to probe the effect of glucose binding on the local and overall structural organization of the protein. The labeling of the N-terminus with a fluorescence probe as well as the protein intrinsic fluorescence were also used to obtain a complete picture of the GGBP structure and dynamics. Our results showed that the binding of glucose to GGBP resulted in no stabilizing effect on the N-terminus portion of GGBP and in a moderate stabilization of the protein matrix in the vicinity of the ligand-binding site. On the contrary, it was observed that the binding of glucose has a strong stabilization effect on the C-terminal domain of the GGBP structure.

In this work is presented the first attempt to develop a fluorescence assay for detection of traces of gluten in food by utilizing the recombinant glutamine-binding protein (GlnBP) from E. coli. We found that GlnBP specifically binds the sequence of amino acids present both in gliadin and other prolamines classified as toxic for celiac patients. Affinity chromatography experiments together with mass spectrometry experiments demonstrated that GlnBP can bind the following amino acid sequence XXQPQPQQQQQQQQQQQQL. Sequence alignment experiments pointed out that this sequence is exclusively representative of the gliadin and the other prolamines considered toxic for celiac patients. These findings suggest the development of a competitive resonance energy transfer (RET) assay for an easy and rapid detection of this sequence in raw and cooked food.

In this work, the fluorescence of glutamine-binding protein (GlnBP) and its complex with glutamine (GlnBP/Gln) in native and unfolded forms was studied. The experimental data were interpreted on the basis of the results of the analysis of Trp and Tyr microenvironments taking into the account the data for GlnBP mutated forms Trp32Phe(Tyr) and Trp220Phe(Tyr), which have been obtained by Axelsen et al. (Biophys. J. 1991, 60, 650-659). This allowed us to explain the negligible contribution of Tyr residues to the bulk fluorescence of the native protein, the similarity of the fluorescence characteristics of GlnBP and GlnBP/Gln, and the uncommon effect of the excess of the fluorescence intensity at 365 nm (Trp emission) upon excitation at 297 nm respect to the excitation at 280 nm. The last effect is explained by the spectral dependence of the Trp 32 and Trp 220 contributions to the protein absorption. The protein Trp fluorescence dependence on the excitation wavelength must be taken into account for the evaluation of the Tyr residues contribution to the bulk fluorescence of protein, and in principle, it also may be used for the development of an approach for the decomposition of a multicomponent protein fluorescence spectrum.