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Mus m 1.0102 is a member of the mouse Major Urinary Protein family, belonging to the Lipocalins superfamily. Major Urinary Proteins (MUPs) are characterized by highly conserved structural motifs. These include a disulphide bond, involved in protein oxidative folding and protein structure stabilization, and a free cysteine residue, substituted by serine only in the pheromonal protein Darcin (MUP20). The free cysteine is recognized as responsible for the onset of inter- or intramolecular thiol/disulphide exchange, an event that favours protein aggregation. Here we show that the substitution of selected cysteine residues modulates Mus m 1.0102 protein folding, fold stability and unfolding reversibility, while maintaining its allergenic potency. Recombinant allergens used for immunotherapy or employed in allergy diagnostic kits require, as essential features, conformational stability, sample homogeneity and proper immunogenicity. In this perspective, recombinant Mus m 1.0102 might appear reasonably adequate as lead molecule because of its allergenic potential and thermal stability. However, its modest resistance to aggregation renders the protein unsuitable for pharmacological preparations. Point mutation is considered a winning strategy. We report that, among the tested mutants, C138A mutant acquires a structure more resistant to thermal stress and less prone to aggregation, two events that act positively on the protein shelf life. Those features make that MUP variant an attractive lead molecule for the development of a diagnostic kit and/or a vaccine., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The presented data were obtained with the lipocalin allergen Mus m 1.0102 and its cysteine mutants MM-C138A, MM-C157A and MM-C138,157A, whose structural features and unfold reversibility investigations are presented in the research article entitled "The allergen Mus m 1.0102: cysteine residues and molecular allergology" [1]. The data were obtained by means of a Dynamic Light Scattering-based thermal stability assay, a Fluorescence-based ligand-binding assay and a basophil degranulation test, and describe proteins' fold stability, ligand binding ability and allergenic potential, respectively. Analysis of the collected data produced the temperatures corresponding to the onset of the protein unfolding, the dissociation constants for N-Phenyl-1-naphthylamine ligand and the profiles of β-hexosaminidase release from RBL SX-38 cells, sensitized with the serum of selected allergic patients and incubated with increasing antigens concentrations. These data allow for comparison of the lipocalin allergen Mus m 1.0102 with its conserved cysteines mutants and, with regard to their potential application in allergy diagnostics and immunotherapy, they contribute to the process of recombinant allergen characterization and standardization., (© 2020 The Author(s).)

Gelonin from the Indian plant Gelonium multiflorum belongs to the type I ribosome-inactivating proteins (RIPs). Like other members of RIPs, this toxin glycoprotein inhibits protein synthesis of eukaryotic cells; hence, it is largely used in the construction of immunotoxins composed of cell-targeted antibodies. Lysosomal degradation is one of the main issues in targeted tumor therapies, especially for type I RIP-based toxins, as they lack the translocation domains. The result is an attenuated cytosolic delivery and a decrease of the antitumor efficacy of these plant-derived toxins; therefore, strategies to permit their release from endosomal vesicles or modifications of the toxins to make them resistant to degradation are necessary to improve their efficacy. Using infrared spectroscopy, we thoroughly analyzed both the secondary structure and the thermal unfolding of gelonin. Moreover, by the combination of two-dimensional correlation spectroscopy and phase diagram method, it was possible to deduce the sequence of events during the unfolding, confirming the typical characteristic of the RIP members to denature in two steps, as a sequential loss of tertiary and secondary structure was detected at 58 °C and at 65 °C, respectively. Additionally, some discrepancies in the unfolding process between gelonin and saporin-S6, another type I RIP protein, were detected.

Interaction with model phospholipid membranes of lupin seed γ-conglutin, a glycaemia-lowering protein from Lupinus albus seeds, has been studied by means of Fourier-Transform infrared spectroscopy at p 2 H 7.0 and at p 2 H 4.5. The protein maintains the same secondary structure both at p 2 H 7.0 and at p 2 H 4.5, but at p 2 H 7.0 a higher 1 H/ 2 H exchange was observed, indicating a greater solvent accessibility. The difference in T m and T D1/2 of the protein at the abovementioned p 2 H's has been calculated around 20 °C. Infrared measurements have been then performed in the presence of DMPG and DOPA at p 2 H 4.5. DMPG showed a little destabilizing effect while DOPA exerted a great stabilizing effect, increasing the T m of γ-conglutin at p 2 H 4.5 of more than 20 °C. Since γ-conglutin at p 2 H 4.5 is in the monomeric form, the interaction with DOPA likely promotes the oligomerization even at p 2 H 4.5. Interaction between DMPG or DOPA and γ-conglutin has been confirmed by turbidity experiments with DMPC:DMPG or DOPC:DOPA SUVs. Turbidity data also showed high-affinity binding of γ-conglutin to anionic SUVs made up with DOPA. The molecular features outlined in this study are relevant to address the applicative exploitation and to delineate a deeper comprehension of the natural functional role of γ-conglutin., (Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.)

Bovine α1-acid glycoprotein (bAGP), a thermostable counterpart of its human homologue, is a positive acute phase protein involved in binding and transportation of a large number of bio-active molecules and drugs across the body. We have investigated the effect of low pH and reducing conditions on the structure of the protein and found that it aggregates at high temperatures. The aggregates show a fibrillar structure when observed with electron microscopy. Aggregation assays using the amyloid-specific dye Thioflavin T show the presence of a lag phase which was neither abolished nor shortened when seeds were added. A priori reduction of the two disulfide bridges of bAGP, on the other hand, abolished the lag phase and reveals a connection between the kinetics of reduction and aggregation. We provide a kinetic interpretation and the corresponding rate laws allowing to model the process of fibril formation by bAGP under reducing conditions. Our interpretation allows to assess the role of disulfide bridges on the fibrillation kinetics of bAGP and can provide a more accurate interpretation of the fibrillation kinetics of other amyloidogenic proteins containing disulfide bridges., (Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Saporin-S6 is a plant toxin belonging to the type 1 ribosome-inactivating protein (RIP) family. Since it was extracted and isolated from Saponaria officinalis for the first time almost thirty years ago, the protein has been widely studied mainly for its potential applications in anti-tumour and anti-viral infection therapy. Like other RIPs, saporin-S6 is particularly effective in the form of immunotoxin conjugated with monoclonal antibodies and its chemico-physical characteristics made the protein a perfect candidate for the synthesis, development and use of saporin-S6-based chimeric toxins. The high stability of the protein against different denaturing agents has been broadly demonstrated, however, its complete thermal unfolding characterization has not already been performed. In this work we analyse in detail structure, thermostability and unfolding features by means of infrared spectroscopy coupled with two-dimensional correlation spectroscopy. Our data showed that saporin-S6 in solution at neutral pH exhibits a secondary structure analogue to that of the crystal and confirmed its good stability at moderately high temperatures, with a temperature of melting of 58°C. Our results also demonstrated that the thermal unfolding process is non-cooperative and occurs in two steps, and revealed the sequence of the events that take place during the denaturation, showing a higher stability of the N-terminal domain of the protein., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Bacterial HtrAs are proteases engaged in extracytoplasmic activities during stressful conditions and pathogenesis. A model prokaryotic HtrA (HtrA/DegP from Escherichia coli) requires activation to cleave its substrates efficiently. In the inactive state of the enzyme, one of the regulatory loops, termed LA, forms inhibitory contacts in the area of the active center. Reduction of the disulfide bond located in the middle of LA stimulates HtrA activity in vivo suggesting that this S-S bond may play a regulatory role, although the mechanism of this stimulation is not known. Here, we show that HtrA lacking an S-S bridge cleaved a model peptide substrate more efficiently and exhibited a higher affinity for a protein substrate. An LA loop lacking the disulfide was more exposed to the solvent; hence, at least some of the interactions involving this loop must have been disturbed. The protein without S-S bonds demonstrated lower thermal stability and was more easily converted to a dodecameric active oligomeric form. Thus, the lack of the disulfide within LA affected the stability and the overall structure of the HtrA molecule. In this study, we have also demonstrated that in vitro human thioredoxin 1 is able to reduce HtrA; thus, reduction of HtrA can be performed enzymatically.

α1-Acid glycoprotein (AGP) is a plasma protein and a member of the acute phase response. AGP is known to bind and carry several biologically active compounds, as well as to down-modulate the immune system activities. In this work, the structure of bovine AGP has been investigated by Fourier-Transform infrared spectroscopy. A model structure has been obtained on the basis of human AGP and refined by molecular dynamics. In spite of the similar structure, bovine AGP shows an unexpectedly higher (∼20 °C) thermostability than its human counterpart. Inspection of the model structure has pointed out the presence of 12 ionic bridges and 2 sulphur-aromatic interactions, whereas only 6 ionic bridges were detected in human AGP. The high number (9) of glutamic acid residues involved in the ionic interactions might explain the significantly decreased thermostability measured at pH 5.5 (Tm ∼ 71 °C) with respect to pH 7.4 (Tm ∼ 81 °C), whereas thermostability of human AGP was only slightly affected by lowering the pH. As in human AGP and several other lipocalins, a temperature-induced molten globule state has been observed in the denaturation pathway of bovine AGP., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Human α(1)-acid glycoprotein (AGP) is a positive acute phase plasma protein containing two disulfide bridges. Structural studies have shown that under specific conditions AGP undergoes aggregation. In this study, we analysed the nature of AGP's aggregates formed under reducing and non-reducing conditions at pH 5.5 and at relatively low temperatures. Thioflavin T and Congo red spectroscopic analyses indicated the presence of cross-β structures in both unreduced and reduced AGP aggregates. In these samples amyloid-like fibrils were detected by transmission electron microscopy. The fibrils are branched and bent and present in very large amount in reduced AGP. Kinetics of AGP fibrillation proceeds without a lag phase and the rate constants of cross-β formation are linearly dependent on AGP concentration and result higher under reducing conditions. The data suggest a possible downhill mechanism of polymerization with a first-order monomer concentration dependence. Bioinformatics tools highlighted an extended region that sheathes one side of the molecule containing aggregation-prone regions. Reducing conditions make the extended region less constricted, allowing greater exposure of aggregation-prone regions, thus explaining the higher propensity of AGP to aggregate and fibrillate., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Thymoquinone (TQ) is the main bioactive component isolated from Nigella sativa essential oil and seeds and has been used for the treatment of inflammations, liver disorders, arthritis, and is of great importance as a promising therapeutic drug for different diseases including cancer. This paper reports the first experimental evidence on binding of TQ to human α(1)-acid glycoprotein (AGP), an important drug-binding glycoprotein in human plasma, which affects pharmacokinetic properties of various therapeutic agents. The interaction of TQ with AGP has been characterized by Fourier transform infrared (FTIR) and fluorescence spectroscopy, as well as by molecular docking experiments. FTIR spectroscopy showed that the binding of TQ to AGP slightly increases its thermal stability and shifts the existence of a molten globule-like state observed in a previous study to higher temperature. The binding constants K(a); the number of binding sites n; and the corresponding thermodynamic parameters ΔG, ΔH, and ΔS at different temperatures were calculated through fluorescence spectroscopy. Fluorescence quenching experiments indicated that TQ binding involves hydrophobic interactions and to a lower extent hydrogen bonds, in agreement with molecular docking experiments. The data on binding ability of TQ to AGP represent basic information for the TQ pharmacokinetics such as drug metabolism and distribution in the body., (Copyright © 2012 Wiley Periodicals, Inc.)

D-serine dehydratase from Saccharomyces cerevisae is a recently discovered dimeric enzyme catalyzing the β-elimination of D-serine to pyruvate and ammonia. The reaction is highly enantioselective and depends on cofactor pyridoxal-5'-phosphate (PLP) and Zn(2+). In our work, the aldimine linkage tethering PLP to recombinant, tagged D-serine dehydratase (Dsd) has been reduced by treatment with NaBH(4) so as to yield an inactive form of the holoenzyme (DsdR), which was further treated with a protease in order to remove the amino-terminal purification tag. Fourier Transform infrared (FT-IR) spectroscopic analysis revealed that both the reduced form (DsdR) and the reduced/detagged form (DsdRD) maintain the overall secondary structure of Dsd, but featured a significant increased thermal stability. The observed T(m) values for DsdR and for DsdRD shifted to 71.5 °C and 73.3 °C, respectively, resulting in nearly 11 °C and 13 °C higher than the one measured for Dsd. Furthermore, the analysis of the FT-IR spectra acquired in the presence of D-serine and L-serine indicates that, though catalytically inert, DsdRD retains the ability to enantioselectively bind its natural substrate. Sequence analysis of D-serine dehydratase and other PLP-dependent enzymes also highlighted critical residues involved in PLP binding. In virtue of its intrinsic properties, DsdRD represents an ideal candidate for the design of novel platforms based on stable, non-consuming binding proteins aimed at measuring d-serine levels in biological fluids., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

Human α(1)-acid glycoprotein (AGP) is an acute phase plasma glycoprotein containing two disulfide bridges. As a member of the lipocalin superfamily, it binds and transports several basic and neutral ligands, but a number of other activities have also been described. Thanks to its binding properties, AGP is also a good candidate for the development of biosensors and affinity chromatography media, and in this context detailed structural information is needed. The structural properties of AGP at different p(2)Hs and under reducing conditions were analysed by FT-IR spectroscopy. The obtained data indicate that AGP, when denatured, does not aggregate at neutral or basic p(2)Hs whilst it does at acidic p(2)Hs. Under reducing conditions the protein is remarkably less thermostable than its oxidized counterpart and presents an enhanced tendency to aggregate, even at neutral p(2)H. A heat-induced molten globule-like state (MG) was detected at 55 °C at p(2)H 7.4 and 5.5. At p(2)H 4.5 the MG occurred at 45 °C with an onset of formation at 40 °C. The MG was not observed under reducing conditions. A lower affinity of chlorpromazine and progesterone for the MG formed at p(2)H 4.5 and 40 °C was observed, suggesting that ligand(s) may be released near the negative surfaces of biological membranes. Furthermore, the reduced AGP displays an enhanced affinity for progesterone, indicating the importance of disulfide bonds for the binding capacity of AGP., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

In Nigeria, Mucuna pruriens seeds are locally prescribed as an oral prophylactic for snake bite and it is claimed that when two seeds are swallowed they protect the individual for a year against snake bites. In order to understand the Mucuna pruriens antisnake properties, the proteins from the acqueous extract of seeds were purified by three chromatographic steps: ConA affinity chromatography, tandem anionic-cationic exchange and gel filtration, obtaining a fraction conventionally called gpMucB. This purified fraction was analysed by SDS-PAGE obtaining 3 bands with apparent masses ranging from 20 to 24 kDa, and by MALDI-TOF which showed two main peaks of 21 and 23 kDa and another small peak of 19 kDa. On the other hand, gel filtration analysis of the native protein indicated a molecular mass of about 70 kDa suggesting that in its native form, gpMucB is most likely an oligomeric multiform protein. Infrared spectroscopy of gpMucB indicated that the protein is particularly thermostable both at neutral and acidic pHs and that it is an all beta protein. All data suggest that gpMucB belongs to the Kunitz-type trypsin inhibitor family explaining the direct anti-snake venom activity of Mucuna pruriens seeds., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

D-serine dehydratase (Dsd) from baker's yeast is a recently discovered enzyme catalyzing the oxidation of D-serine to pyruvate and ammonia. The reaction depends on the cofactors pyridoxal-5'-phosphate (PLP) and Zn(2+), featuring a very high selectivity towards the D-enantiomer of the amino acid serine. In humans, altered levels of D-serine in the cerebrospinal fluid (CSF) and blood correlate with the onset and evolution of a number of neurodegenerative diseases. Up to date very little is known on the structure of Dsd. Hence, we have investigated the structure of this enzyme by means of Fourier Transform infrared (FT-IR) spectroscopy and used the structural data derived thereof to validate a homology model of Dsd. In this model, Dsd adopts a fold that is characteristic of type III pyridoxal-dependent enzymes. This consists of an α/β (TIM) barrel and a β-sandwich domain at the N- and C-termini, respectively. Analysis of the Amide I and Amide III infrared bands revealed that the amounts of α (24%), β (29%) and unordered structures (47%) correlate well with those derived from the model (25%, 29% and 46% respectively), suggesting reliability of the latter. In addition, the model of Dsd was further refined by recreating the PLP- and zinc-restored active site based on a PLP- and zinc-dependent bacterial amino acid racemase recently crystallized, allowing us to identify the potential cofactor and metal binding residues of Dsd., (Copyright © 2010 Elsevier Masson SAS. All rights reserved.)

Protein disulfide oxidoreductases (PDOs) are ubiquitous redox enzymes that catalyse dithiol-disulfide exchange reactions. PDOs have been well studied in bacteria and eukarya, and they have been described in a number of thermophilic and hyperthermophilic species, where they play a critical role in the structural stabilization of intracellular proteins. In this study, the effect of high hydrostatic pressure on the structural properties of PDO from the hyperthermophilic archaeon Pyrococcus furiosus (PfPDO) was analysed in order to gain insights on the possible mechanisms used to endure extreme environmental conditions. The protein is highly thermostable and the data indicate that PfPDO is highly piezostable as well, and that different areas of the protein have a different local compressibility and resistance to high hydrostatic pressure. In particular, the results show that alpha-helices are more sensitive to pressure up to 5 kbar, whilst within 5-9 kbar the loss of beta-sheets is more pronounced than the loss of alpha-helices. Examination of the PfPDO structure and calculations of the solvent accessible surface areas for each amino acid indicate that 42% of the PfPDO residues are buried and that the protein contains four small internal hydrophobic cavities. These findings are discussed in terms of important factors contributing to the high piezostability and thermostability of the enzyme.

The aim of this study was to characterize the role of particular elements of the regulatory loop L2 in the activation process and maintaining the proteolytic activity of HtrA (DegP) from Escherichia coli. We measured the effects of various mutations introduced to the L2 loop's region (residues 228-238) on the stability of HtrA molecule and its proteolytic activity. We demonstrated that most mutations affected the activity of HtrA. In the case of the following substitutions: L229N, N235I, I238N, the proteolytic activity was undetectable. Thus, the majority of interactions mediated by the studied amino-acid residues seem to play important role in maintaining the active conformation. Formation of contacts between the apical parts (residues 231-234) of the L2 loops within the HtrA trimer, in particular the residues D232, was shown to play a crucial role in the activation process of HtrA. Stabilization of these intermolecular interactions by substitution of D232 with valine caused a stimulation of proteolytic activity whereas deletion of this region abolished the activity. Since the pathogenic E. coli strains require active HtrA for virulence, the apical part of L2 is of particular interest in terms of structure-based drug design for treatment E. coli infections., (2010 Elsevier Inc. All rights reserved.)

Thymoquinone (TQ) is the main constituent of Nigella sativa essential oil which shows promising in vitro and in vivo antineoplastic growth inhibition against various tumor cell lines. Because of the increasing interest to test it in pre-clinical and clinical researches for assessing its health benefits, we here evaluate the interactions between TQ and human serum albumin (HSA), a possible carrier of this drug in vivo. Binding to HSA was studied using different spectroscopic techniques. Fourier transform infrared (FT-IR) and circular dichroism (CD) spectroscopies suggest that the association between TQ and HSA does not affect the secondary structure of HSA. Using fluorescence spectroscopy, one mole of TQ was found to bind one mole of HSA with a binding constant of 2.39 +/- 0.2 10(4)M(-1). At 25 degrees C (pH 7.4), van't Hoff's enthalpy and entropy that accompany the binding were found to be -10.24 kJ/mol(-1) and 45 J/mol(-1)K(-1) respectively. The thermodynamic analysis of the TQ-HSA complex formation shows that the binding process is enthalpy driven and spontaneous, and that hydrophobic interactions are the predominant intermolecular forces stabilizing the complex. Furthermore, displacement experiments using warfarin and ibuprofen indicate that TQ could bind to site I of HSA, which is also in agreement with the results of the molecular modeling study., (2010 Elsevier GmbH. All rights reserved.)

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.

The present investigation was undertaken to characterize mechanism of thermal activation of serine protease HtrA (DegP) from Escherichia coli. We monitored the temperature-induced structural changes within the regulatory loops L1, L2 and LA using a set of single-Trp HtrA mutants. The accessibility of each Trp residue to aqueous medium at temperature range 25-45 degrees C was assessed by steady-state fluorescence quenching using acrylamide and these results in combination with mean fluorescence lifetimes (tau) and wavelength emission maxima (lambda(em)max) were correlated with the induction of the HtrA proteolytic activity. Generally the temperature shift caused better exposure of Trps to the quencher; although, each of the loops was affected differently. The LA loop seemed to be the most prone to temperature-induced conformational changes and a significant opening of its structure was observed even at the lowest temperatures tested (25-30 degrees C). To the contrary, the L1 loop, containing the active site serine, remained relatively unchanged up to 40 degrees C. The L2 loop was the most exposed element and showed the most pronounced changes at temperatures exceeding 35 degrees C. Summing up, the HtrA structure appears to open gradually, parallel to the gradual increase of its proteolytic activity.

The effect of temperature on the structure of the rat odorant-binding protein was investigated by spectroscopic and in silico methodologies. In particular, in this work, we examined the structural features of the rat OBP-1F by Fourier-transform infrared spectroscopy and molecular dynamics investigations. The obtained spectroscopic results were analyzed using the following three different methods based on the unexchanged amide hydrogens of the protein sample: (1) the analysis of difference spectra; (2) the generalized 2D-IR correlation spectroscopy; (3) the phase diagram method. The three methods indicated that at high temperatures the rOBP-1F structure undergoes a relaxation process involving the protein tertiary organization before undergoing the denaturation and aggregation processes, suggesting the presence of an intermediate state such as a molten globule-like state. Importantly, the proposed analyses represent a general approach that could be applied to the study of protein stability.

The exploration of events taking place at different timescales and affecting the structural and dynamics properties of proteins, such as the interactions of proteins with ligands and the subunits association/ dissociation, must necessarily be performed by using different methodologies, each of which specialized to highlight the different phenomena that occur when proteins are exposed to chemical or physical stress. In this work, we investigated the structure and dynamics of the wild-type bovine odorant-binding protein (wt-bOBP), which is a domain-swapped dimeric protein, and the triple mutant deswapped monomeric form of the protein (m-bOBP) to shed light on the role of the quaternary and tertiary structural organization in the protein thermal stability. Difference infrared spectra, 2D-IR correlation spectroscopy and molecular dynamics simulations were used to probe the effect of heating on protein structure and dynamics in microsecond and nanoseconds temporal ranges, respectively. The obtained results show that there is a heating-induced transition toward a less structured state in m-bOBP, that it is detectable around 70-80 degrees C. On the contrary, in wt-bOBP this transition is almost negligible, and changes are detectable in the protein spectra in the range of temperature between 75 and 85 degrees C. A detailed 3D inspection of the structure of the two proteins that takes into the account the spectroscopic results indicates that (a) ion pairs and hydrophobic interactions appear to be the major determinants responsible for the protein stability and (b) the protein intersubunit interactions confer an increased resistance toward the thermal stress.

The trehalose/maltose-binding protein (MalE1) is one component of trehalose and maltose uptake system in the thermophilic organism Thermus thermophilus. MalE1 is a monomeric 48 kDa protein predominantly organized in alpha-helix conformation with a minor content of beta-structure. In this work, we used Fourier-infrared spectroscopy and in silico methodologies for investigating the structural stability properties of MalE1. The protein was studied in the absence and in the presence of maltose as well as in the absence and in the presence of SDS at different p(2)H values (neutral p(2)H and at p(2)H 9.8). In the absence of SDS, the results pointed out a high thermostability of the MalE1 alpha-helices, maintained also at basic p(2)H values. However, the obtained data also showed that at high temperatures the MalE1 beta-sheets underwent to structural rearrangements that were totally reversible when the temperature was lowered. At room temperature, the addition of SDS to the protein solution slightly modified the MalE1 secondary structure content by decreasing the protein thermostability. The infrared data, corroborated by molecular dynamics simulation experiments performed on the structure of MalE1, indicated that the protein hydrophobic interactions have an important role in the MalE1 high thermostability. Finally, the results obtained on MalE1 are also discussed in comparison with the data on similar thermostable proteins already studied in our laboratories.

Fourier-transform infrared spectroscopy, in vitro bioassay and enzyme-linked immunoassay were used to study the structural-functional relationships of recombinant mink growth hormone (mGH), refolded and stored under different conditions. Porcine GH (pGH) was synthesized and used as an example. These two hormones, when refolded and stored the same way, had the same secondary structures, biological and immunological efficacy, and biological potency. Only the immunological potency differed, mGH being significantly less potent than pGH. Renaturation pH and storing frozen or at 4 degrees C in 5% glycerol did not affect either the secondary structure or the activity. However, freeze-drying raised the content of buried alpha-helices and lowered that of solvated alpha-helices and of unordered structures. These conformational changes were associated with a reduction of immunological and biological potency of mGH and of immunological potency of pGH. These findings provide original information on the secondary structure of mGH, and show that conformational changes induced by lyophilization adversely affect its activity.

Reactive oxygen species generated upon UV-A exposure appear to play a major role in dermal connective tissue transformations including degradation of skin collagen. Here we investigate on oxidative damage to collagen achieved by exposure to (i) UV-A irradiation and to (ii) AAPH-derived radicals and on its possible prevention using synthetic and natural antioxidants. Oxidative damage was identified through SDS-PAGE, circular dichroism spectroscopy and quantification of protein carbonyl residues. Collagen (2 mg/ml) exposed to UV-A and to AAPH-derived radicals was degraded in a time- and dose-dependent manner. Upon UV-A exposure, maximum damage was observable at 730 kJ/m2 UV-A, found to be equivalent to roughly 2 h of sunshine, while exposure to 5 mM AAPH for 2 h at 50 degrees C lead to maximum collagen degradation. In both cases, dose-dependent protection was achieved by incubation with muM concentrations of nitroxide radicals, where the extent of protection was shown to be dictated by their structural differences whereas the vitamins E and C proved less efficient inhibitors of collagen damage. These results suggest that nitroxide radicals may be able to prevent oxidative injury to dermal tissues in vivo alternatively to commonly used natural antioxidants.

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.

The identification of the determinants of protein thermal stabilization is often pursued by comparing enzymes from hyperthermophiles with their mesophilic counterparts while direct structural comparisons among proteins and enzymes from hyperthermophiles are rather uncommon. Here, oligomeric beta-glycosidases from the hyperthermophilic archaea Sulfolobus solfataricus (Ss beta-gly), Thermosphaera aggregans (Ta beta-gly), and Pyrococcus furiosus (Pf beta-gly), have been compared. Studies of FTIR spectroscopy and kinetics of thermal inactivation showed that the three enzymes had similar secondary structure composition, but Ss beta-gly and Ta beta-gly (temperatures of melting 98.1 and 98.4 degrees C, respectively) were less stable than Pf beta-gly, which maintained its secondary structure even at 99.5 degrees C. The thermal denaturation of Pf beta-gly, followed in the presence of SDS, suggested that this enzyme is stabilized by hydrophobic interactions. A detailed inspection of the 3D-structures of these enzymes supported the experimental results: Ss beta-gly and Ta beta-gly are stabilized by a combination of ion-pairs networks and intrasubunit S-S bridges while the increased stability of Pf beta-gly resides in a more compact protein core. The different strategies of protein stabilization give experimental support to recent theories on thermophilic adaptation and suggest that different stabilization strategies could have been adopted among archaea., (2007 Wiley-Liss, Inc.)

Hsp70 (DnaK) is a highly conserved molecular chaperone present in bacteria, eukaryotes, and some archaea. In a previous work we demonstrated that DnaK from the archaeon Methanosarcina mazei (DnaK(Mm)) and the DnaK from the bacterium Escherichia coli (DnaK(Ec)) were functionally similar when assayed in vitro but DnaK(Mm) failed to substitute for DnaK(Ec) in vivo. Searching for the molecular basis of the observed DnaK species specificity we compared substrate binding by DnaK(Mm) and DnaK(Ec). DnaK(Mm) showed a lower affinity for the model peptide (a-CALLQSRLLS) compared to DnaK(Ec). Furthermore, it was unable to negatively regulate the E. coli sigma32 transcription factor level under heat shock conditions and poorly bound purified sigma32, which is a native substrate of DnaK(Ec). These observations taken together indicate differences in substrate specificity of archaeal and bacterial DnaKs. Structural modeling of DnaK(Mm) showed some structural differences in the substrate-binding domains of DnaK(Mm) and DnaK(Ec), which may be responsible, at least partially, for the differences in peptide binding. Size-exclusion chromatography and native gel electrophoresis revealed that DnaK(Mm) was found preferably in high molecular mass oligomeric forms, contrary to DnaK(Ec). Oligomers of DnaK(Mm) could be dissociated in the presence of ATP and a substrate (peptide) but not ADP, which may suggest that monomer is the active form of DnaK(Mm).

Hsp70s are chaperone proteins that are conserved in evolution and present in all prokaryotic and eukaryotic organisms. In the archaea, which form a distinct kingdom, the Hsp70 chaperones have been found in some species only, including Methanosarcina mazei. Both the bacterial and archaeal Hsp70(DnaK) chaperones cooperate with a GrpE co-chaperone which stimulates the ATPase activity of the DnaK protein. It is currently believed that the archaeal Hsp70 system was obtained by the lateral transfer of chaperone genes from bacteria. Our previous finding that the DnaK and GrpE proteins of M. mazei can functionally cooperate with the Escherichia coli GrpE and DnaK supported this hypothesis. However, the cooperation was surprising, considering the very low identity of the GrpE proteins (26%) and the relatively low identity of the DnaK proteins (56%). The aim of this work was to investigate the molecular basis of the observed interspecies chaperone interaction. Infrared resolution-enhanced spectra of the M. mazei and E. coli DnaK proteins were almost identical, indicating high similarity of their secondary structures, however, some small differences in band position and in the intensity of amide I' band components were observed and discussed. Profiles of thermal denaturation of both proteins were similar, although they indicated a higher thermostability of the M. mazei DnaK compared to the E. coli DnaK. Electrophoresis under non-denaturing conditions demonstrated that purified DnaK and GrpE of E. coli and M. mazei formed mixed complexes. Protein modeling revealed high similarity of the 3-dimensional structures of the archaeal and bacterial DnaK and GrpE proteins.

The effect of the pressure on the structure and stability of the D-galactose/D-glucose binding protein from Escherichia coli in the absence (GGBP) and in the presence (GGBP/Glc) of glucose was studied by Fourier transform infrared (FT-IR) spectroscopy and molecular dynamic (MD) simulations. FT-IR spectroscopy experiments showed that the protein beta-structures are more resistant than alpha-helices structures to pressure value increases. In addition, the infrared data indicated that the binding of glucose stabilizes the protein structure against high pressure values, and the protein structure does not completely unfold up to pressure values close to 9000 bar. MD simulations allow a prediction of the most probable configuration of the protein, consistent with the increasing pressures on the two systems. The detailed analysis of the structures at molecular level confirms that, among secondary structures, alpha-helices are more sensitive than beta-structures to the destabilizing effect of high pressure and that glucose is able to preserve the structure of the protein in the complex. Moreover, the evidence of the different resistance of the two domains of this protein to high pressure is investigated and explained at a molecular level, indicating the importance of aromatic amino acid in protein stabilization.

We have previously shown that the major ion-pairs network of the tetrameric beta-glycosidase from the hyperthermophilic archeon Sulfolobus solfataricus involves more than 16 ion-pairs and hydrogen bonds between several residues from the four subunits and protects the protein from thermal unfolding by sewing the carboxy-termini of the enzyme. We show here that the amino-terminal of the enzyme also plays a relevant role in the thermostabilization of the protein. In fact, the addition of four extra amino acids at the amino-terminal of the beta-glycosidase, though not affecting the catalytic machinery of the enzyme and its thermophilicity, produced a faster enzyme inactivation in the temperature range 85-95 degrees C and decreased the Tm of the protein of 6 degrees C, measured by infrared spectroscopy. In addition, detailed two-dimensional IR correlation analysis revealed that the quaternary structure of the tagged enzyme is destabilized at 85 degrees C whilst that of the wild type enzyme is stable up to 98 degrees C. Molecular models allowed the rationalization of the experimental data indicating that the longer amino-terminal tail may destabilize the beta-glycosidase by enhancing the molecular fraying of the polypeptide and loosening the dimeric interfaces. The data support the hypothesis that fraying of the polypeptide chain termini is a relevant event in protein unfolding.

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 degrees-100 degrees C. The results show that TMBP possesses remarkable temperature stability and its secondary structure does not melt up to 90 degrees 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 degrees 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 alpha-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., (2006 Wiley-Liss, Inc.)

The abundant zinc finger proteins (ZFPs) sharing the KRAB motif, a potent transcription repression domain, direct the assembly on templates of multiprotein repression complexes. A pivotal step in this pathway is the assembly of a KRAB domain-directed complex with a primary corepressor, KAP1/KRIP-1/TIF1beta. The structure/function dependence of KRAB/TIF1beta protein-protein interaction and properties of the complex, therefore, play pivotal roles in diverse cellular processes depending on KRAB-ZFPs regulation. KRAB domains are functionally bipartite. The 42 amino acid-long KRAB-A module, indeed, is necessary and sufficient for transcriptional repression and for the interaction with the tripartite RBCC region of TIF1beta, while the KRAB-B motif seems to potentiate the assembly of the complex. The structural properties of KRAB-A and KRAB-AB domains from the human ZNF2 protein have been investigated by characterizing highly purified lone (A) and composite (AB) modules. Hydrodynamic and spectroscopic features, investigated by means of gel filtration, circular dichroism, and infrared spectroscopy, provide evidence that both KRAB-A and KRAB-AB domains present low compactness, structural disorder, residual secondary structure content, flexibility, and tendency to molecular aggregation. Comparative analysis among KRAB-A and KRAB-AB modules suggests that the presence of the -B module may influence the properties of lone KRAB-A by affecting the structural flexibility and stability of the conformers. The combined experimental data and the intrinsic features of KRAB-A and KRAB-AB primary structures indicate a potential role of specific subregions within the modules in driving structural flexibility, which is proposed to be of importance for their function.

The effect of the depletion of calcium on the structure and thermal stability of the D-galactose/D-glucose-binding protein (GGBP) from Escherichia coli was studied by fluorescence spectroscopy and Fourier-transform infrared spectroscopy. The calcium-depleted protein (GGBP-Ca) was also studied in the presence of glucose (GGBP-Ca/Glc). The results show that calcium depletion has a small effect on the secondary structure of GGBP, and, in particular it affects a population of alpha-helices with a low exposure to solvent. Alternatively, glucose-binding to GGBP-Ca eliminates the effect induced by calcium depletion by restoring a secondary structure similar to that of the native protein. In addition, the infrared and fluorescence data obtained reveal that calcium depletion markedly reduces the thermal stability of GGBP. In particular, the spectroscopic experiments show that the depletion of calcium mainly affects the stability of the C-terminal domain of the protein. However, the binding of glucose restores the thermal stability of GGBP-Ca. The thermostability of GGBP and GGBP-Ca was also studied by molecular dynamics simulations. The simulation data support the spectroscopic results. New insights into the role of calcium in the thermal stability of GGBP contribute to a better understanding of the protein function and constitute important information for the development of biotechnological applications of this protein. Mutations and/or labelling of amino acid residues located in the protein C-terminal domain may affect the stability of the whole protein structure.

Despite extensive investigations on thermal denaturation of alpha(1)-acid glycoprotein (AGP) using a variety of techniques, structural features of the folded-unfolded state in terms of residual secondary structures and the structural transitions involved in this process have not been fully characterized. In this study we employed FT-IR spectroscopy to investigate the thermal unfolding and reversibility of temperature-induced changes in AGP. The data revealed a fully reversible beta-sheet-rich protein which exhibits a molten globule-like state, an important protein folding intermediate. 2D-IR COS revealed the sequence of the conformational changes occurring before denaturation and confirmed the formation of this intermediate which was further supported by CD spectroscopy. On account of the similarities in the FT-IR spectra of AGP with those of porcine odorant-binding protein (OBP), homology modeling of AGP using OBP as template was performed. The resemblance of AGP and OBP 3D structures confirmed the similarities of data obtained using FT-IR spectroscopy. Overall, FT-IR spectroscopy appears to be useful for investigating the structural characteristics and stability of proteins whose 3D structures are unavailable and for assessing the molten globule-like state in small beta-sheet-rich proteins.

The effect of SDS, pD, and temperature on the structure and stability of the protein disulfide oxidoreductase from Pyrococcus furiosus (PfPDO) was investigated by molecular dynamic (MD) simulations and FT-IR spectroscopy. pD affects the thermostability of alpha-helices and beta-sheets differently, and 0.5% or higher SDS concentration influences the structure significantly. The experiments allowed us to detect a secondary structural reorganization at a definite temperature and pD which may correlate with a high ATPase activity of the protein. The MD simulations supported the infrared data and revealed the different behavior of the N and C terminal segments, as well as of the two active sites.

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 alpha-helices than beta-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., ((c) 2004 Wiley-Liss, Inc.)

Beta-glycosidase from the hyperthermophilic archaeon Sulfolobus solfataricus is a homotetramer with a higher number of ion pairs compared with mesophilic glycoside hydrolases. The ion pairs are arranged in large networks located mainly at the tetrameric interface of the molecule. In the present study, the structure and thermal stability of the wild-type beta-glycosidase and of three mutants in residues R488 and H489 involved in the C-terminal ionic network were examined by FTIR (Fourier-transform IR) spectroscopy. The FTIR data revealed small differences in the secondary structure of the proteins and showed a lower thermostability of the mutant proteins with respect to the wild-type. Generalized 2D-IR (two-dimensional IR correlation spectroscopy) at different temperatures showed different sequences of thermal unfolding events in the mutants with respect to the wild-type, indicating that punctual mutations affect the unfolding and aggregation process of the protein. A detailed 2D-IR analysis of synchronous maps of the proteins allowed us to identify the temperatures at which the ionic network that stabilizes the quaternary structure of the native and mutant enzymes at the C-terminal breaks down. This evidence gives support to the current theories on the mechanism of ion-pair stabilization in proteins from hyperthermophilic organisms.

In steroid hydroxylation system in adrenal cortex mitochondria, NADPH-adrenodoxin reductase (AR) and adrenodoxin (Adx) form a short electron-transport chain that transfers electrons from NADPH to cytochromes P-450 through FAD in AR and [2Fe-2S] cluster in Adx. The formation of [AR/Adx] complex is essential for the electron transfer mechanism in which previous studies suggested that AR tryptophan (Trp) residue(s) might be implicated. In this study, we modified AR Trps by N-bromosuccinimide (NBS) and studied AR binding to Adx by a resonant mirror biosensor. Chemical modification of tryptophans caused inhibition of electron transport. The modified protein (AR*) retained the native secondary structure but showed a lower affinity towards Adx with respect to AR. Activity measurements and fluorescence data indicated that one Trp residue of AR may be involved in the electron transferring activity of the protein. Computational analysis of AR and [AR/Adx] complex structures suggested that Trp193 and Trp420 are the residues with the highest probability to undergo NBS-modification. In particular, the modification of Trp420 hampers the correct reorientation of AR* molecule necessary to form the native [AR/Adx] complex that is catalytically essential for electron transfer from FAD in AR to [2Fe-2S] cluster in Adx. The data support an incorrect assembly of [AR*/Adx] complex as the cause of electron transport inhibition., (Copyright 2004 Wiley-Liss, Inc.)

Methylation in vivo is a post-translational modification observed in several organisms belonging to eucarya, bacteria, and archaea. Although important implications of this modification have been demonstrated in several eucaryotes, its biological role in hyperthermophilic archaea is far from being understood. The aim of this work is to clarify some effects of methylation on the properties of beta-glycosidase from Sulfolobus solfataricus, by a structural comparison between the native, methylated protein and its unmethylated counterpart, recombinantly expressed in Escherichia coli. Analysis by Fourier transform infrared spectroscopy indicated similar secondary structure contents for the two forms of the protein. However, the study of temperature perturbation by Fourier transform infrared spectroscopy and turbidimetry evidenced denaturation and aggregation events more pronounced in recombinant than in native beta-glycosidase. Red Nile fluorescence analysis revealed significant differences of surface hydrophobicity between the two forms of the protein. Unlike the native enzyme, which dissociated into SDS-resistant dimers upon exposure to the detergent, the recombinant enzyme partially dissociated into monomers. By electrospray mapping, the methylation sites of the native protein were identified. A computational analysis of beta-glycosidase three-dimensional structure and comparisons with other proteins from S. solfataricus revealed analogies in the localization of methylation sites in terms of secondary structural elements and overall topology. These observations suggest a role for the methylation of lysyl residues, located in selected domains, in the thermal stabilization of beta-glycosidase from S. solfataricus.

We have examined and compared the effects of mutating Y41 and H155 in the iron superoxide dismutase (SOD) from the archaeon Sulfolobus solfataricus (Ss). These two neighboring residues in the active site are known to have crucial functions in structurally related SODs from different sources. The metal analysis indicates a slightly lower iron content after either Y41F or H155Q replacement, without any significant substitution of iron for manganese. The specific activity of SsSOD referred to the iron content is 17-fold reduced in the Y41F mutant, whereas it is less than 2-fold reduced by the H155Q mutation. The noticeable pH dependence of the activity of SsSOD and H155Q-SsSOD, due to the ionization of Y41 (pK 8.4), is lost in Y41F-SsSOD. After H155Q and even more after the Y41F substitution, the archaeal enzyme acquires a moderate sensitivity to sodium azide inhibition. The hydrogen peroxide inactivation of SsSOD is significantly increased after H155Q replacement; however, both mutants are insensitive to the modification of residue 41 by phenylmethanesulfonyl fluoride. Heat inactivation studies showed that the high stability of SsSOD is reduced by the H155Q mutation; however, upon the addition of SDS, a much faster inactivation kinetics is observed both with wild-type and mutant SsSOD forms. The detergent is also required to follow thermal denaturation of the archaeal enzyme by Fourier transform infrared spectroscopy; these studies gave information about the effect of mutations and modification on flexibility and compactness of the protein structure. The crystal structure of Y41F mutant revealed an uninterrupted hydrogen bond network including three solvent molecules connecting the iron-ligating hydroxide ion via H155 with F41 and H37, which is not present in structures of the corresponding mutant SODs from other sources. These data suggest that Y41 and H155 are important for the structural and functional properties of SsSOD; in particular, Y41 seems to be a powerful regulator of the activity of SsSOD, whereas H155 is apparently involved in the organization of the active site of the enzyme.