Maria Staiano

Proteins from extremophiles as stable tools for advanced biotechnological applications of high social interest

Extremophiles are micro-organisms adapted to survive in ecological niches defined as ‘extreme’ for humans and characterized by the presence of adverse environmental conditions, such as high or low temperatures, extreme values of pH, high salt concentrations or high pressure. Biomolecules isolated from extremophiles possess extraordinary properties and, in particular, proteins isolated from extremophiles represent unique biomolecules that function under severe conditions, comparable to those prevailing in various industrial processes. In this article, we will review some examples of recent applications of thermophilic proteins for the development of a new class of fluorescence non-consuming substrate biosensors for monitoring the levels of two analytes of high social interest, such as glucose and sodium.

The protein scaffold of the lipocalin odorant-binding protein is suitable for the design of new biosensors for the detection of explosive components

The detection of hazard exposure is a current priority, including the detection of traces of explosive molecules in different environments like luggage storage rooms and public places, and is becoming a major requirement for homeland security. In the present study we carried out a preliminary investigation on the binding capacities of four forms of the lipocalin odorant-binding protein (OBP) for the detection of explosive components such as diphenylamine, dimethyl-phthalate, resorcinol and dinitrotoluene. The experimental results, showing that OBP binds these compounds with affinity constants ranging between 80 nM and 10.6 mM, indicate that this protein can be used as a probe for the realization of a biosensor to sense explosive compounds.

Nanostructured Silver-Based Surfaces: New Emergent Methodologies for an Easy Detection of Analytes

In this work, we describe how to realize a new sensing platform for an easy and fast detection of analytes. In particular, we utilized enhanced fluorescence emission on silver island films (SIFs) coupled to the total internal reflection fluorescence mode (TIRF) to develop a new assay format for the detection of target analytes. Here, as an example, we report on the detection of the toxic peptides present in gliadin (Gli). Our assay was performed as follows: (1) gliadin was first captured on surfaces coated with anti-Gli antibodies; (2) the surfaces were then incubated with fluorophore-labeled anti-Gli antibodies; (3) the signal from the fluorophore-labeled anti-Gli antibody bound to the antigen was detected by TIRF. The system was examined on glass surfaces and on SIFs. We observed a relevant enhancement of the signal from SIFs compared to the signal from the glass substrate not modified with a SIF. In addition, the estimated detection limit (EDL) of our methodology was 60 ng/mL (or lower). This limit is therefore lower than the clinical cut-off for Gli presence in food for celiac patients. The advantage of our method is a reduced number of testing steps, which allows for easy detection of residual toxic peptides in food labeled as gluten free. The proposed technology can be easily expanded to the determination of different target analytes.

Hydrophobic interactions and ionic networks play an important role in thermal stability and denaturation mechanism of the porcine odorant-binding protein.

Despite the fact that the porcine odorant‐binding protein (pOBP) possesses a single tryptophan residue (Trp 16) that is characterized by a high density microenvironment (80 atoms in a sphere with radius 7 Å) with only one polar group (Lys 120) and three bound water molecules, pOBP displayed a red shifted fluorescence emission spectrum (λmax = 340 nm). The protein unfolding in 5M GdnHCl was accompanied by the red shift of the fluorescence emission spectrum (λmax = 353 nm), by the increase of fluorescence quantum yield, and by the decrease of lifetime of the excited state (from 4.25 ns in native state to 3.15 ns in the presence of 5M GdnHCl). Taken together these data indicate the existence of an exciplex complex (Trp 16 with Lys 120 and/or with bound molecules of water) in the protein native state. Heat‐induced denaturation of pOBP resulted in significant red shifts of the fluorescence emission spectra: the value of the ratio (I320/I365) upon excitation at λex = 297 nm (parameter A) decreases from 1.07 to 0.64 passing from 60 to 85°C, and the calculated midpoint of transition was centered at 70°C. Interestingly, even at higher temperature, the values of the parameter A both in the absence and in the presence of GdnHCl did not coincide. This suggests that a portion of the protein structure is still preserved upon the temperature‐induced denaturation of the protein in the absence of GdnHCl. CD experiments performed on pOBP in the absence and in the presence of GdnHCl and at different temperatures were in agreement with the fluorescence results. In addition, the obtained experimental data were corroborated by the analysis of the 3D structure of pOBP which revealed the amino acid residues that contribute to the protein dynamics and stability. Finally, molecular dynamics simulation experiments pointed out the important role of ion pair interactions as well as the molecular motifs that are responsible for the high thermal stability of pOBP, and elucidated the reasons of the protein aggregation that occurred at high temperature. Proteins 2008.

Binding of glutamine to glutamine-binding protein from Escherichia coli induces changes in protein structure and increases protein stability

Glutamine‐binding protein (GlnBP) from Escherichia coli is a monomeric protein localized in the periplasmic space of the bacterium. It is responsible for the first step in the active transport of L‐glutamine across the cytoplasmic membrane. The protein consists of two similar globular domains linked by two peptide hinges, and X‐ray crystallographic data indicate that the two domains undergo large movements upon ligand binding. Fourier transform infrared spectroscopy (FTIR) was used to analyze the structure and thermal stability of the protein in detail. The data indicate that glutamine binding induces small changes in the secondary structure of the protein and that it renders the structure more thermostable and less flexible. Detailed analyses of IR spectra show a lower thermal sensitivity of α‐helices than β‐sheets in the protein both in the absence and in the presence of glutamine. Generalized two‐dimensional (2D) analyses of IR spectra reveal the same sequence of unfolding events in the protein in the absence and in the presence of glutamine, indicating that the amino acid does not affect the unfolding pathway of the protein. The data give new insight into the structural characteristics of GlnBP that are useful for both basic knowledge and biotechnological applications. Proteins 2005.

The role of calcium in the conformational dynamics and thermal stability of the D-galactose/D-glucose-binding protein from Escherichia coli

We have characterized stability and conformational dynamics of the calcium depleted D‐galactose/D‐glucose‐binding protein (GGBP) from Escherichia coli. The structural stability of the protein was investigated by steady state and time resolved fluorescence, and far‐UV circular dichroism in the temperature range from 20°C to 70°C. We have found that the absence of the Ca2+ ion results in a significant destabilization of the C‐terminal domain of the protein. In particular, the melting temperature decreases by about 10°C with the simultaneous loss of the melting cooperativity. Time resolved fluorescence quenching revealed significant loosening of the protein when highly shielded Trp residue(s) became accessible to acrylamide at higher temperatures. We have documented a significant stabilizing effect of glucose that mostly reverts the effect of calcium, that is, the thermal stability of the protein increases by about 10°C and the melting cooperativity is restored. Moreover, the protein structure remains compact with low amplitude of the segmental mobility up to high temperatures. We have used molecular dynamics to identify the structural feature responsible for changes in the temperature stability. Disintegration of the Ca2+‐binding loop seems to be responsible for the loss of the stability in the absence of calcium. The new insights on the structural properties and temperature stability of the calcium depleted GGBP contribute to better understanding of the protein function and constitute important information for the development of new biotechnological applications of this class of proteins. Proteins 2005.

Structural and thermal stability characterization of Escherichia coli D-galactose/D-glucose-binding protein.

The effect of temperature and glucose binding on the structure of the galactose/glucose‐binding protein from Escherichia coli was investigated by circular dichroism, Fourier transform infrared spectroscopy, and steady‐state and time‐resolved fluorescence. The data showed that the glucose binding induces a moderate change of the secondary structure content of the protein and increases the protein thermal stability. The infrared spectroscopy data showed that some protein stretches, involved in α‐helices and β strand conformations, are particularly sensitive to temperature. The fluorescence studies showed that the intrinsic tryptophanyl fluorescence of the protein is well represented by a three‐exponential model and that in the presence of glucose the protein adopts a structure less accessible to the solvent. The new insights on the structural properties of the galactose/glucose‐binding protein can contribute to a better understanding of the protein functions and represent fundamental information for the development of biotechnological applications of the protein.

D-trehalose/D-maltose-binding protein from the hyperthermophilic archaeon Thermococcus litoralis : The binding of trehalose and maltose results in different protein conformational states

In this work, we used fluorescence spectroscopy, molecular dynamics simulation, and Fourier transform infrared spectroscopy for investigating the effect of trehalose binding and maltose binding on the structural properties and the physical parameters of the recombinant D‐trehalose/D‐maltose binding protein (TMBP) from the hyperthermophilic archaeon Thermococcus litoralis. The binding of the two sugars to TMBP was studied in the temperature range 20°–100°C. The results show that TMBP possesses remarkable temperature stability and its secondary structure does not melt up to 90°C. Although both the secondary structure itself and the sequence of melting events were not significantly affected by the sugar binding, the protein assumes different conformations with different physical properties depending whether maltose or trehalose is bound to the protein. At low and moderate temperatures, TMBP possesses a structure that is highly compact both in the absence and in the presence of two sugars. At about 90°C, the structure of the unliganded TMBP partially relaxes whereas both the TMBP/maltose and the TMBP/trehalose complexes remain in the compact state. In addition, Fourier transform infrared results show that the population of α‐helices exposed to the solvent was smaller in the absence than in the presence of the two sugars. The spectroscopic results are supported by molecular dynamics simulations. Our data on dynamics and stability of TMBP can contribute to a better understanding of transport‐related functions of TMBP and constitute ground for targeted modifications of this protein for potential biotechnological applications. Proteins 2006.

A Loose Domain Swapping Organization Confers a Remarkable Stability to the Dimeric Structure of the Arginine Binding Protein from Thermotoga maritima

The arginine binding protein from Thermatoga maritima (TmArgBP), a substrate binding protein (SBP) involved in the ABC system of solute transport, presents a number of remarkable properties. These include an extraordinary stability to temperature and chemical denaturants and the tendency to form multimeric structures, an uncommon feature among SBPs involved in solute transport. Here we report a biophysical and structural characterization of the TmArgBP dimer. Our data indicate that the dimer of the protein is endowed with a remarkable stability since its full dissociation requires high temperature as well as SDS and urea at high concentrations. In order to elucidate the atomic level structural properties of this intriguing protein, we determined the crystallographic structures of the apo and the arginine-bound forms of TmArgBP using MAD and SAD methods, respectively. The comparison of the liganded and unliganded models demonstrates that TmArgBP tertiary structure undergoes a very large structural re-organization upon arginine binding. This transition follows the Venus Fly-trap mechanism, although the entity of the re-organization observed in TmArgBP is larger than that observed in homologous proteins. Intriguingly, TmArgBP dimerizes through the swapping of the C-terminal helix. This dimer is stabilized exclusively by the interactions established by the swapping helix. Therefore, the TmArgBP dimer combines a high level of stability and conformational freedom. The structure of the TmArgBP dimer represents an uncommon example of large tertiary structure variations amplified at quaternary structure level by domain swapping. Although the biological relevance of the dimer needs further assessments, molecular modelling suggests that the two TmArgBP subunits may simultaneously interact with two distinct ABC transporters. Moreover, the present protein structures provide some clues about the determinants of the extraordinary stability of the biomolecule. The availability of an accurate 3D model represents a powerful tool for the design of new TmArgBP suited for biotechnological applications.

Amino acid transport in thermophiles: characterization of an arginine-binding protein in Thermotoga maritima.2.Molecular organization and structural stability

ABC transport systems provide selective passage of metabolites across cell membranes and typically require the presence of a soluble binding protein with high specificity to a specific ligand. In addition to their primary role in nutrient gathering, the binding proteins associated with bacterial transport systems have been studied for their potential to serve as design scaffolds for the development of fluorescent protein biosensors. In this work, we used Fourier transform infrared spectroscopy and molecular dynamics simulations to investigate the physicochemical properties of a hyperthermophilic binding protein from Thermotoga maritima. We demonstrated preferential binding for the polar amino acid arginine and experimentally monitored the significant stabilization achieved upon binding of ligand to protein. The effect of temperature, pH, and detergent was also studied to provide a more complete picture of the protein dynamics. A protein structure model was obtained and molecular dynamic experiments were performed to investigate and couple the spectroscopic observations with specific secondary structural elements. The data determined the presence of a buried beta-sheet providing significant stability to the protein under all conditions investigated. The specific amino acid residues responsible for arginine binding were also identified. Our data on dynamics and stability will contribute to our understanding of bacterial binding protein family members and their potential biotechnological applications.