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In this paper, we report, for the first time to our knowledge, a new versatile and future proof diagnostic platform for diverse applications and heterogeneous biological targets. This platform relies on a plasmonic-augmented silicon photonic biochip to detect bacteria and protein biomarkers within minutes. To demonstrate the potential of this platform in diverse applications, we demonstrate the detection of two heterogeneous targets, the bacterium Escherichia coli (E. coli) and the molecule C-reactive protein (CRP) using a universal detection method. E. coli is one of the most commonly encountered bacterial pathogens involved in food monitoring, food born infections and water contamination applications, while CRP is a well-established disease severity indicator frequently used in common clinical practice. The biochip used is fully compatible with CMOS semiconductor manufacturing, while it hosts biosensors arrays for any combination of detection assays. Each biosensor exploits a 70 μm long aluminum plasmonic transducer within a silicon nitride waveguide-based Mach–Zehnder Interferometer. Each aluminum surface was silanized and biomodified with specific antibodies. Biosensing experiments revealed that CRP can be detected in diverse sample mediums, and E. coli detection was achieved in buffer at concentrations as low as 10 cells/ml within 13–25 min. The results are in good agreement with preceding numerical simulations. The modular nature of the reported biosensing platform makes it scalable and customizable, allowing nearly any combination of diagnostic tests targeting pathogens and proteins to be integrated on the same biochip for food quality monitoring, environmental monitoring, drug discovery and modern cell therapy manufacturing, or biomedical applications.

Cyanobacteria bloom is the term used to describe an abnormal and rapid growth of cyanobacteria in aquatic ecosystems such as lakes, rivers, and oceans as a consequence of anthropic factors, ecosystem degradation, or climate change. Cyanobacteria belonging to the genera Microcystis, Anabaena, Planktothrix, and Nostoc produce and release toxins called microcystins (MCs) into the water. MCs can have severe effects on human and animal health following their ingestion and inhalation. The MC structure is composed of a constant region (composed of five amino acid residues) and a variable region (composed of two amino acid residues). When the MC variable region is composed of arginine and leucine, it is named MC-LR. The most-common methods used to detect the presence of MC-LR in water are chromatographic-based methods (HPLC, LC/MS, GC/MS) and immunological-based methods (ELISA). In this work, we developed a new competitive Förster resonance energy transfer (FRET) assay to detect the presence of traces of MC-LR in water. Monoclonal antibody anti-MC-LR and MC-LR conjugated with bovine serum albumin (BSA) were labeled with the near-infrared fluorophores CF568 and CF647, respectively. Steady-state fluorescence measurements were performed to investigate the energy transfer process between anti-MC-LR 568 and MC-LR BSA 647 upon their interaction. Since the presence of unlabeled MC-LR competes with the labeled one, a lower efficiency of FRET process can be observed in the presence of an increasing amount of unlabeled MC-LR. The limit of detection (LoD) of the FRET assay is found to be 0.245 nM (0.245 µg/L). This value is lower than the provisional limit established by the World Health Organization (WHO) for quantifying the presence of MC-LR in drinking water.

The development of sensitive methods for the detection of endotoxin molecules, such as lipopolysaccharides (LPS), is essential for food safety and health control. Conventional analytical methods used for LPS detection are based on the pyrogen test, plating and culture-based methods, and the limulus amoebocyte lysate method (LAL). Alternatively, the development of reliable biosensors for LPS detection would be highly desirable to solve some critical issues, such as high cost and a long turnaround time. In this work, we present a label-free Surface-Enhanced Raman Spectroscopy (SERS)-based method for LPS detection in its free form. The proposed method combines the benefits of plasmonic enhancement with the selectivity provided by a specific anti-lipid A antibody (Ab). A high-enhancing nanostructured silver substrate was coated with Ab. The presence of LPS was quantitatively monitored by analyzing the changes in the Ab spectra obtained in the absence and presence of LPS. A limit of detection (LOD) and quantification (LOQ) of 12 ng/mL and 41 ng/mL were estimated, respectively. Importantly, the proposed technology could be easily expanded for the determination of other biological macromolecules.

Food allergies are an exceptional response of the immune system caused by the ingestion of specific foods. The main foods responsible for allergic reactions are milk, eggs, seafood, soy, peanuts, tree nuts, wheat, and their derived products. Chicken egg ovalbumin (OVA), a common allergen molecule, is often used for the clarification process of wine. Traces of OVA remain in the wine during the fining process, and they can cause significant allergic reactions in sensitive consumers. Consequently, the European Food Safety Authority (EFSA) and the American Food and Drug Administration (FDA) have shown the risks for allergic people to assume allergenic foods and food ingredients, including eggs. Commonly, OVA detection requires sophisticated and time-consuming analytical techniques. Intending to develop a faster assay, we designed a proof-of-concept non-Faradaic impedimetric immunosensor for monitoring the presence of OVA in wine. Polyclonal antibodies anti-OVA were covalently immobilised onto an 11-mercaptoundecanoic-acid (11-MUA)-modified gold surface. The developed immunosensor was able to detect OVA in diluted white wine without the need for an external probe or any pre-treatment step with a sensitivity of 0.20 µg/mL, complying with the limit established by the resolution OIV/COMEX 502–2012 for the quantification of allergens in wine.

The demand for a wide choice of food that is safe and palatable increases every day. Consumers do not accept off-flavors that have atypical odors resulting from internal deterioration or contamination by substances alien to the food. Odor response depends on the volatile organic compounds (VOCs), and their detection can provide information about food quality. Gas chromatography/mass spectrometry is the most powerful method available for the detection of VOC. However, it is laborious, costly, and requires the presence of a trained operator. To develop a faster analytic tool, we designed a non-Faradaic impedimetric biosensor for monitoring the presence of VOCs involved in food spoilage. The biosensor is based on the use of the pig odorant-binding protein (pOBP) as the molecular recognition element. We evaluated the affinity of pOBP for three different volatile organic compounds (1-octen-3-ol, trans-2-hexen-1-ol, and hexanal) related to food spoilage. We developed an electrochemical biosensor conducting impedimetric measurements in liquid and air samples. The impedance changes allowed us to detect each VOC sample at a minimum concentration of 0.1 μM.

Abstract Listeria monocytogenes is a foodborne pathogen responsible for human listeriosis. The increasing incidence of listeriosis induced governments and food manufacturing enterprises to act to diminish the problem. Several methods for the detection of Listeria monocytogenes in food industries were developed. However, they are time-consuming and require the use of specialized equipment. To reduce the detection time of Listeria monocytogenes in food, in this work we developed a fluorescence sandwich immunoassay based on the use of an innovative chitosan-cellulose nanocrystal (CNC) membrane that improves the antigen capture during bacterial growth. The combined use of CNC film for the capture of p60 protein-specific antigen together with the use of fluorescence detection reduced the time of analysis from 24 to 12 h with a limit of detection (LOD) of the assay of 102 CFU/mL (2 Log). In addition, the use of monoclonal anti-PepD covalently immobilized to a CNC membrane assured a high specificity of the assay. Interestingly, the obtained results show no cross-reactivity with the five most diffused pathogen bacteria strains tested.

Volatile organic compounds (VOCs) from ‘emitting’ plants inform the ‘receiving’ (listening) plants of impending stresses or simply of their presence. However, the receptors that allow receivers to detect the volatile cue are elusive. Most likely, plants (as animals) have odorant-binding proteins (OBPs), and in fact, a few OBPs are known to bind ‘stress-induced’ plant VOCs. We investigated whether these and other putative OBPs may bind volatile constitutive and stress-induced isoprenoids, the most emitted plant VOCs, with well-established roles in plant communication and defense. Molecular docking simulation experiments suggest that structural features of a few plant proteins screened in databases could allow VOC binding. In particular, our results show that monoterpenes may bind the same plant proteins that were described to bind other stress-induced VOCs, while the constitutive hemiterpene isoprene is unlikely to bind any investigated putative OBP and may not have an info-chemical role. We conclude that, as for animal, there may be plant OBPs that bind multiple VOCs. Plant OBPs may play an important role in allowing plants to eavesdrop messages by neighboring plants, triggering defensive responses and communication with other organisms.

The enzyme nicotinate phosphoribosyltransferase (NAPRT) mediates the rate-limiting step in NAD salvage pathway starting from nicotinic acid. Here the authors show that NAPRT can be detected extracellularly, binds to Toll like receptor 4, and activates NF-kB signaling and cytokine production in macrophage via NAD synthesis-independent pathways.

Abstract Background Shp1, a tyrosine-phosphatase-1 containing the Src-homology 2 (SH2) domain, is involved in inflammatory and immune reactions, where it regulates diverse signalling pathways, usually by limiting cell responses through dephosphorylation of target molecules. Moreover, Shp1 regulates actin dynamics. One Shp1 target is Src, which controls many cellular functions including actin dynamics. Src has been previously shown to be activated by a signalling cascade initiated by the cytosolic-phospholipase A2 (cPLA2) metabolite glycerophosphoinositol 4-phosphate (GroPIns4P), which enhances actin polymerisation and motility. While the signalling cascade downstream Src has been fully defined, the mechanism by which GroPIns4P activates Src remains unknown. Methods Affinity chromatography, mass spectrometry and co-immunoprecipitation studies were employed to identify the GroPIns4P-interactors; among these Shp1 was selected for further analysis. The specific Shp1 residues interacting with GroPIns4P were revealed by NMR and validated by site-directed mutagenesis and biophysical methods such as circular dichroism, isothermal calorimetry, fluorescence spectroscopy, surface plasmon resonance and computational modelling. Morphological and motility assays were performed in NIH3T3 fibroblasts. Results We find that Shp1 is the direct cellular target of GroPIns4P. GroPIns4P directly binds to the Shp1-SH2 domain region (with the crucial residues being Ser 118, Arg 138 and Ser 140) and thereby promotes the association between Shp1 and Src, and the dephosphorylation of the Src-inhibitory phosphotyrosine in position 530, resulting in Src activation. As a consequence, fibroblast cells exposed to GroPIns4P show significantly enhanced wound healing capability, indicating that GroPIns4P has a stimulatory role to activate fibroblast migration. GroPIns4P is produced by cPLA2 upon stimulation by diverse receptors, including the EGF receptor. Indeed, endogenously-produced GroPIns4P was shown to mediate the EGF-induced cell motility. Conclusions This study identifies a so-far undescribed mechanism of Shp1/Src modulation that promotes cell motility and that is dependent on the cPLA2 metabolite GroPIns4P. We show that GroPIns4P is required for EGF-induced fibroblast migration and that it is part of a cPLA2/GroPIns4P/Shp1/Src cascade that might have broad implications for studies of immune-inflammatory response and cancer.

African swine fever (ASF) is one of the most dangerous hemorrhagic infectious diseases that affect domestic and wild pigs. Currently, neither a vaccine nor effective treatments are available for this disease. As regards the degree of virulence, ASFV strains can be divided into high, moderate, or low virulence. The main detection methods are based on the use of the polymerase chain reaction (PCR). In order to prevent an uncontrolled spread of ASF, new on-site techniques that can enable the identification of an early-stage disease are needed. We have developed a specific immunological SPR-based assay for ASFV antigen detection directly in liquid samples. The developed assay allows us to detect the presence of ASFV at the dose of 103 HAD50/mL.

Odorant-binding proteins (OBPs) are a group of small and soluble proteins present in both vertebrates and insects. They have a high level of structural stability and bind to a large spectrum of odorant molecules. In the environmental field, benzene is the most dangerous compound among the class of pollutants named BTEX (benzene, toluene, ethylbenzene, and xylene). It has several effects on human health and, consequently, it appears to be important to monitor its presence in the environment. Commonly, its detection requires the use of very sophisticated and time-consuming analytical techniques (GC-MS, etc.) as well as the presence of specialized personnel. Here, we present the application of an odorant-binding protein (pOBP) isolated from pigs as a molecular recognition element (MRE) for a low-energy impedenziometric biosensor for outdoor and real-time benzene detection. The obtained results show that the biosensor can detect the presence of 64 pM (5 µg/m3) benzene, the limit value of exposure for human health set by the European Directive 2008/50/EC.

In this paper we present the development of photonic integrated circuit (PIC) biosensors for the label-free detection of six emerging and endemic swine viruses, namely: African Swine Fever Virus (ASFV), Classical Swine Fever Virus (CSFV), Porcine Reproductive and Respiratory Syndrome Virus (PPRSV), Porcine Parvovirus (PPV), Porcine Circovirus 2 (PCV2), and Swine Influenza Virus A (SIV). The optical biosensors are based on evanescent wave technology and, in particular, on Resonant Rings (RRs) fabricated in silicon nitride. The novel biosensors were packaged in an integrated sensing cartridge that included a microfluidic channel for buffer/sample delivery and an optical fiber array for the optical operation of the PICs. Antibodies were used as molecular recognition elements (MREs) and were selected based on western blotting and ELISA experiments to ensure the high sensitivity and specificity of the novel sensors. MREs were immobilized on RR surfaces to capture viral antigens. Antibody–antigen interactions were transduced via the RRs to a measurable resonant shift. Cell culture supernatants for all of the targeted viruses were used to validate the biosensors. Resonant shift responses were dose-dependent. The results were obtained within the framework of the SWINOSTICS project, contributing to cover the need of the novel diagnostic tools to tackle swine viral diseases.

Nicotinamide mononucleotide (NMN) is a key intermediate in the nicotinamide adenine dinucleotide (NAD+) biosynthesis. Its supplementation has demonstrated beneficial effects on several diseases. The aim of this study was to characterize NMN deamidase (PncC) inactive mutants to use as possible molecular recognition elements (MREs) for an NMN-specific biosensor. Thermal stability assays and steady-state fluorescence spectroscopy measurements were used to study the binding of NMN and related metabolites (NaMN, Na, Nam, NR, NAD, NADP, and NaAD) to the PncC mutated variants. In particular, the S29A PncC and K61Q PncC variant forms were selected since they still preserve the ability to bind NMN in the micromolar range, but they are not able to catalyze the enzymatic reaction. While S29A PncC shows a similar affinity also for NaMN (the product of the PncC catalyzed reaction), K61Q PncC does not interact significantly with it. Thus, PncC K61Q mutant seems to be a promising candidate to use as specific probe for an NMN biosensor.

The purpose of this work is to provide an exhaustive overview of the emerging biosensor technologies for the detection of analytes of interest for food, environment, security, and health. Over the years, biosensors have acquired increasing importance in a wide range of applications due to synergistic studies of various scientific disciplines, determining their great commercial potential and revealing how nanotechnology and biotechnology can be strictly connected. In the present scenario, biosensors have increased their detection limit and sensitivity unthinkable until a few years ago. The most widely used biosensors are optical-based devices such as surface plasmon resonance (SPR)-based biosensors and fluorescence-based biosensors. Here, we will review them by highlighting how the progress in their design and development could impact our daily life.

The synthesis of two 5′-end (4-dimethylamino)azobenzene conjugated G-quadruplex forming aptamers, the thrombin binding aptamer (TBA) and the HIV-1 integrase aptamer (T30695), was performed. Their structural behavior was investigated by means of UV, CD, fluorescence spectroscopy, and gel electrophoresis techniques in K+-containing buffers and water-ethanol blends. Particularly, we observed that the presence of the 5′-(4-dimethylamino)azobenzene moiety leads TBA to form multimers instead of the typical monomolecular chair-like G-quadruplex and almost hampers T30695 G-quadruplex monomers to dimerize. Fluorescence studies evidenced that both the conjugated G-quadruplexes possess unique fluorescence features when excited at wavelengths corresponding to the UV absorption of the conjugated moiety. Furthermore, a preliminary investigation of the trans-cis conversion of the dye incorporated at the 5′-end of TBA and T30695 showed that, unlike the free dye, in K+-containing water-ethanol-triethylamine blend the trans-to-cis conversion was almost undetectable by means of a standard UV spectrophotometer.

In recent years, air pollution has been a subject of great scientific and public interests for the strong impact on human health. Air pollution is due to the presence in the atmosphere of polluting substances, such as carbon monoxide, sulfur and nitrogen oxides, particulates and volatile organic compounds (VOCs), derived predominantly from various combustion processes. Benzene is a VOC belonging to group-I carcinogens with a toxicity widely demonstrated. The emission limit values and the daily exposure time to benzene (TLV-TWA) are 5μg/m3 (0.00157 ppm) and 1.6mg/m3 (0.5 ppm), respectively. Currently, expensive and time-consuming analytical methods are used for detection of benzene. These methods require to perform a few preliminary steps such as sampling, and matrices pre-treatments. In addition, it is also needed the support of specialized personnel. Recently, single-walled carbon nanotube (SWNTs) gas sensors with a limit detection (LOD) of 20 ppm were developed for benzene detection. Other innovative bioassay, called bio-report systems, were proposed. They use a whole cell (Pseudomona putida or Escherichia coli) as molecular recognition element and exhibit a LOD of about 10 μM. Here, we report on the design of a highly sensitive fluorescence assay for monitoring atmospheric level of benzene. For this purpose, we used as molecular recognition element the porcine odorant-binding protein (pOBP). 1-Aminoanthracene was selected as extrinsic fluorescence probe for designing a competitive fluorescence resonance energy transfer (FRET) assay for benzene detection. The detection limit of our assay was 3.9μg/m3, a value lower than the actual emission limit value of benzene as regulated by European law.

Abstract Filamentous bacteriophage fd particles delivering antigenic determinants via DEC‐205 (fdsc‐αDEC) represent a powerful delivery system that induces CD8+ T‐cell responses even when administered in the absence of adjuvants or maturation stimuli for dendritic cells. In order to investigate the mechanisms of this activity, RNA‐Sequencing of fd‐pulsed dendritic cells was performed. A significant differential expression of genes involved in innate immunity, co‐stimulation and cytokine production was observed. In agreement with these findings, we demonstrate that induction of proinflammatory cytokines and type I interferon by fdsc‐αDEC was MYD88 mediated and TLR9 dependent. We also found that fdsc‐αDEC is delivered into LAMP‐1‐positive compartments and co‐localizes with TLR9. Thus, phage particles containing a single‐strand DNA genome rich in CpG motifs delivered via DEC‐205 are able to intercept and trigger the active TLR9 innate immune receptor into late endosome/lysosomes and to enhance the immunogenicity of the displayed antigenic determinants. These findings make fd bacteriophage a valuable tool for immunization without administering exogenous adjuvants.

In this paper we introduce a field diagnostic device based on the combination of advanced bio-sensing and photonics technologies, to tackle emerging and endemic viruses causing swine epidemics, and consequently significant economic damage in farms. The device is based on the use of microring resonators fabricated in silicon nitride with CMOS compatible techniques. In the paper, the designed and fabricated photonic integrated circuit (PIC) sensors are presented and characterized, showing an optimized performance in terms of optical losses (30 dB per ring) and extinction ration for ring resonances (15 dB). Furthermore, the results of an experiment for porcine circovirus 2 (PCV2) detection by using the developed biosensors are presented. Positive detection for different virus concentrations has been obtained. The device is currently under development in the framework of the EU Commission co-funded project SWINOSTICS.

In this paper, we present the concept of a novel diagnostic device for on-site analyses, based on the use of advanced bio-sensing and photonics technologies to tackle emerging and endemic viruses causing swine epidemics and significant economic damage in farms. The device is currently under development in the framework of the EU Commission co-funded project. The overall concept behind the project is to develop a method for an early and fast on field detection of selected swine viruses by non-specialized personnel. The technology is able to detect pathogens in different types of biological samples, such as oral fluids, faeces, blood or nasal swabs. The device will allow for an immediate on-site threat assessment. In this work, we present the overall concept of the device, its architecture with the technical requirements, and all the used innovative technologies that contribute to the advancements of the current state of the art.

In this paper, we present WaterSpy, a project developing an innovative, compact, cost-effective photonic device for pervasive water quality sensing, operating in the mid-IR spectral range. The approach combines the use of advanced Quantum Cascade Lasers (QCLs) employing the Vernier effect, used as light source, with novel, fibre-coupled, fast and sensitive Higher Operation Temperature (HOT) photodetectors, used as sensors. These will be complemented by optimised laser driving and detector electronics, laser modulation and signal conditioning technologies. The paper presents the WaterSpy concept, the requirements elicited, the preliminary architecture design of the device, the use cases in which it will be validated, while highlighting the innovative technologies that contribute to the advancement of the current state of the art.

Arginine-binding protein from the extremophile Thermotoga maritima is a 27.7 kDa protein possessing the typical two-domain structure of the periplasmic binding proteins family. The protein is characterized by a very high specificity and affinity to bind to arginine, also at high temperatures. Due to its features, this protein could be taken into account as a potential candidate for the design of a biosensor for arginine. It is important to investigate the stability of proteins when they are used for biotechnological applications. In this article, we review the structural and functional features of an arginine-binding protein from the extremophile Thermotoga maritima with a particular eye on its potential biotechnological applications.

Antibiotics, such as benzyl-penicillin (PenG) and cephalosporin, are the most common compounds used in animal therapy. Their massive and illegal use in animal therapy and prophylaxis inevitably causes the presence of traces in foods of animal origin (milk and meat), which creates several problems for human health. With the aim to prevent the negative impact of β-lactam and, in particular, PenG residues present in the milk on customer health, many countries have established maximum residue limits (MRLs). To cope with this problem here, we propose an effective alternative, compared to the analytical methods actually employed, to quantify the presence of penicillin G using the surface plasmon resonance (SPR) method. In particular, the PenG molecule was conjugated to a protein carrier to immunize a rabbit and produce polyclonal antibodies (anti-PenG). The produced antibodies were used as molecular recognition elements for the design of a competitive immune-assay for the detection of PenG by SPR experiments. The detection limit of the developed assay was found to be 8.0 pM, a value much lower than the MRL of the EU regulation limit that is fixed at 12 nM. Thus, our results clearly show that this system could be successfully suitable for the accurate and easy determination of PenG.

The final goal of this work is to achieve a selective detection of butanal by the realization of a simple, small-size and low cost experimental approach. To this end, a porcine odorant-binding protein was used in connection with surface plasmon resonance transduction in a plastic optical fiber tool for the selective detection of butanal by a competitive assay. This allows to reduce the cost and the size of the sensing device and it offers the possibility to design a "Lab-on-a-chip" platform. The obtained results showed that this system approach is able to selectively detect the presence of butanal in the concentration range from 20 μM to 1000 μM.

The ability of d-glucose/d-galactose-binding protein (GGBP) to reversibly interact with its ligands, glucose and galactose, makes this protein an attractive candidate for sensing elements of glucose biosensors. This potential is largely responsible for attracting researchers to study the conformational properties of this protein. Previously, we showed that an increase in the fluorescence intensity of the fluorescent dye 6-bromoacetyl-2-dimetylaminonaphtalene (BADAN) is linked to the holo-form of the GGBP/H152C mutant in solutions containing sub-denaturing concentrations of guanidine hydrochloride (GdnHCl). It was hypothesized that low GdnHCl concentrations might lead to compaction of the protein, thereby facilitating ligand binding. In this work, we utilize BADAN fluorescence spectroscopy, intrinsic protein UV fluorescence spectroscopy, and isothermal titration calorimetry (ITC) to show that the sub-denaturing GdnHCl concentrations possess osmolyte-like stabilizing effects on the structural dynamics, conformational stability, and functional activity of GGBP/H152C and the wild type of this protein (wtGGBP). Our data are consistent with the model where low GdnHCl concentrations promote a shift in the dynamic distribution of the protein molecules toward a conformational ensemble enriched in molecules with a tighter structure and a more closed conformation. This promotes the increase in the configurational complementarity between the protein and glucose molecules that leads to the increase in glucose affinity in both GGBP/H152C and wtGGBP.

A large group of odorant-binding proteins (OBPs) has attracted great scientific interest as promising building blocks in constructing optical biosensors for dangerous substances, such as toxic and explosive molecules. Native tissue-extracted bovine OBP (bOBP) has a unique dimer folding pattern that involves crossing the α-helical domain in each monomer over the other monomer's β-barrel. In contrast, recombinant bOBP maintaining the high level of stability inherent to native tissue bOBP is produced in a stable native-like state with a decreased tendency for dimerization and is a mixture of monomers and dimers in a buffered solution. This work is focused on the study of the quaternary structure and the folding-unfolding processes of the recombinant bOBP in the absence and in the presence of guanidine hydrochloride (GdnHCl). Our results show that the recombinant bOBP native dimer is only formed at elevated GdnHCl concentrations (1.5 M). This process requires re-organizing the protein structure by progressing through the formation of an intermediate state. The bOBP dimerization process appears to be irreversible and it occurs before the protein unfolds. Though the observed structural changes for recombinant bOBP at pre-denaturing GdnHCl concentrations show a local character and the overall protein structure is maintained, such changes should be considered where the protein is used as a sensitive element in a biosensor system.

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.

In this work, we used a combination of fluorescence correlation spectroscopy (FCS) and molecular dynamics (MD) simulation methodologies to acquire structural information on pH-induced unfolding of the maltotriose-binding protein from Thermus thermophilus (MalE2). FCS has emerged as a powerful technique for characterizing the dynamics of molecules and it is, in fact, used to study molecular diffusion on timescale of microsecond and longer. Our results showed that keeping temperature constant, the protein diffusion coefficient decreased from 84±4 µm(2)/s to 44±3 µm(2)/s when pH was changed from 7.0 to 4.0. An even more marked decrease of the MalE2 diffusion coefficient (31±3 µm(2)/s) was registered when pH was raised from 7.0 to 10.0. According to the size of MalE2 (a monomeric protein with a molecular weight of 43 kDa) as well as of its globular native shape, the values of 44 µm(2)/s and 31 µm(2)/s could be ascribed to deformations of the protein structure, which enhances its propensity to form aggregates at extreme pH values. The obtained fluorescence correlation data, corroborated by circular dichroism, fluorescence emission and light-scattering experiments, are discussed together with the MD simulations results.

In this work we characterize an alcohol dehydrogenase (ADH) from the hyperthermophilic archaeon Pyrobaculum aerophilum (PyAeADHII). We have previously found that PyAeADHII has no activity when standard ADH substrates are used but is active when α-tetralone is used as substrate. Here, to gain insights into enzyme function, we screened several chemical libraries for enzymatic modulators using an assay employing α-tetralone. The results indicate that PyAeADHII activity in the presence of α-tetralone was inhibited by compounds such as flunarizine. We also examined metal coordination of the enzyme in solution by performing metal substitution of the enzyme-bound zinc (Zn²⁺) with cobalt. The solution-based absorption spectra for cobalt substituted PyAeADHII supports substitution at the structural Zn²⁺ site. To gain structural insight, we obtained the crystal structure of both wild-type and cobalt-substituted PyAeADHII at 1.75 Å and 2.20 Å resolution, respectively. The X-ray data confirmed one metal ion per monomer present only at the structural site with otherwise close conservation to other ADH enzymes. We next determined the co-crystal structure of the NADPH-bound form of the enzyme at 2.35 Å resolution to help define the active site region of the enzyme and this data shows close structural conservation with horse ADH, despite the lack of a catalytic Zn²⁺ ion in PyAeADHII. Modeling of α-tetralone into the NADPH bound structure suggests an arginine as a possible catalytic residue. The data presented here can yield a better understanding of alcohol dehydrogenases lacking the catalytic zinc as well as the structural features inherent to thermostable enzymes.

The lipocalin family is typically composed of small proteins characterized by a range of different molecular recognition properties. Odorant binding proteins (OBPs) are a class of proteins of this family devoted to the transport of small hydrophobic molecules in the nasal mucosa of vertebrates. Among OBPs, bovine OBP (bOBP) is of great interest for its peculiar structural organization, characterized by a domain swapping of its two monomeric subunits. The effect of pressure on unfolding and refolding of native dimeric bOBP and of an engineered monomeric form has been investigated by theoretical and experimental studies under pressure. A coherent model explains the pressure-induced protein structural changes: i) the substrate-bound protein stays in its native configuration up to 330 MPa, where it loses its substrate; ii) the substrate-free protein dissociates into monomers at 200 MPa; and iii) the monomeric substrate-free form unfolds at 120 MPa. Molecular dynamics simulations showed that the pressure-induced tertiary structural changes that accompany the quaternary structural changes are mainly localized at the interface between the monomers. Interestingly, pressure-induced unfolding is reversible, but dimerization and substrate binding can no longer occur. The volume of the unfolding kinetic transition state of the monomer has been found to be similar to that of the folded state. This suggests that its refolding requires relatively large structural and/or hydrational changes, explaining thus the relatively low stability of the monomeric form of this class of proteins.