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Substrate-binding proteins (SBPs), members of a protein superfamily, are suitable to be used as molecular recognition elements (MRE) for biosensor development (Dwyer & Hellinga 2004). Their main function is to transport various biomolecules such as sugars, amino acids, peptides and inorganic ions into the cell (Tian et al. 2007). Although these proteins vary in term of size and primary sequence, they have similar tertiary structures. In particular, the three-dimensional structures consist of two domains linked by a hinge region and the ligand-binding site is located at the interface between the two domains (Dattelbaum & Lakowicz 2001). In Gram-positive bacteria SBPs are either anchored to the cytoplasmic membrane or fused to membrane-anchored domains of ATP-binding cassette (ABC) importers (Berntsson et al. 2010). SBPs are highly specific for a wide range of analytes, which bind even in nano-molar concentrations. Moreover SBPs undergo a ligand-induced conformational change that can be detected by variations of optical properties of protein structural features such as fluorescence intensity emission (Grunewald 2014). These properties allow the use of SBPs to construct optical and electrochemical biosensors for a wide variety of chemical classes, including sugars, amino acids, dipeptides, cations and anions. Therefore this class of proteins is widely used as probes for reversible optical sensors of analytes with high social impact. An example of SBP is the Glutamate-binding Protein (GluBP) that is a potential candidate as MRE for a biosensor to detect glutamate. GluBP is a lipoprotein isolated from Corynebacterium glutamicum in which is involved in glutamate uptake. Corynebacterium glutamicum is a Gram-positive, facultative anaerobic, heterotrophic bacterium, which belongs to the mycolic acid-containing actinomycetes. It is not pathogenic and it can be found in soil, animal faeces, fruits and vegetables (Trötschel et al. 2003). Moreover, Corynebacterium glutamicum is very important bacterium involved in monosodium glutamate (MSG) industrial production. It is able to produce large amounts of glutamate, but its uptake carriers allow it to exploit L-glutamate as unique nitrogen and carbon source hence, this bacterium can grow on a minimal medium (Burkovski et al. 2003). Genetic engineering gives the possibility to manipulate genes to modulate the affinity and selectivity of proteins. This allows us to greatly expand the range of biochemically relevant analytes, which can be measured by protein-based sensors. In this context, we studied the possibility to develop a fast and stable glutamate biosensor using Glutamate-binding Protein (GluBP). Recently, crystals of GluBP were obtained but the 3D structure to be resolved by X-Ray crystallography (Liu et al. 2013). In order to expand knowledge on this SBP, the project of thesis is focalized on the functional and structural characterization of GluB-lipoprotein /His-Tag protein (GluB-lP/P). In particular GluB-lP/P from C. glutamicum was deeply studied for the understanding of its structural properties by Fluorescence Spectroscopy and Circular Dichroism. By these techniques, the effect of temperature on the stability of the protein, other chemico-physical perturbation (such as pH and presence of cations) of the intrinsic two fluorescence tryptophan residues were investigated in the absence and in the presence of L-glutamate. Obtained results show that L-glutamate stabilises GluBP secondary and tertiary structures increasing Temperature of melting about 10 °C. Sodium ions increase the thermal stability. Thermal stability increases at pH 8. In addition, L-glutamate binding experiments allowed the calculation of apparent binding Kostant (Kd) of about 6 nM. L-glutamate binding, induces blue-shift in fluorescence spectra from 337 up to 331 nm as happens by reducing the pH from pH 8 to pH 6. Acrylamide fluorescence quenching at pH 8 in the presence of L-glutamate have confirmed that GluBP changes the tertiary structure and tryptophans became buried. In addition GluBP binding experiments were also performed in the presence of L-glutamine and L-aspartate and only in the presence of L-aspartate was possible to obtain a partial increase of thermal stability, which confirmed the specificity of GluBP for L-glutamate. In the near future, the resolution of X-Ray 3D structure of GluBP will allow obtain structural information for GluBP engineering that will allow the development of a fluorescent biosensor against L-glutamate in food, tears, blood sweat and so on.

The PID1/NYGGF4/PCLI1 gene encodes for a protein with a phosphotyrosine-binding domain, which interacts with the lipoprotein receptor-related protein 1. Previous work by us and others suggested a function of the gene in cell proliferation of NIH3T3 fibroblasts and 3T3-L1 pre-adipocytes. The molecular characterization of PCLI1 protein, ectopically expressed in NIH3T3 fibroblasts, revealed two phosphorylation sites at Ser154 and Ser165. In order to clarify the functions of this gene, we analyzed the effects of its downregulation on cellular proliferation and cell cycle progression in NIH3T3 cell cultures. Downregulation of PID1/NYGGF4/PCLI1 mRNA levels by short hairpin RNAs (shRNAs) elicited decreased proliferation rate in mammalian cell lines; cell cycle analysis of serum-starved, synchronized NIH3T3 fibroblasts showed an increased accumulation of shRNA-interfered cells in the G1 phase. Decreased levels of FOS and MYC mRNAs were accordingly associated with these events. The molecular scenario emerging from our data suggests that PID1/NYGGF4/PCLI1 controls cellular proliferation and cell cycle progression in NIH3T3 cells.