Structural analysis of the peptides temporin-Ra and temporin-Rb and interactions with model membranes.

The skin of amphibians is widely exploited as rich sources of membrane active peptides that differ in chain size, polypeptide net charge, secondary structure, target selectivity and toxicity. In this study, two small antimicrobial peptides, temporin-Ra and temporin-Rb, originally isolated from the skin of the European marsh frog (Rana ridibunda), described as active against pathogen bacteria and presenting low toxicity to eukaryotic cells were synthesized and had their physicochemical properties and mechanism of action investigated. The temporin peptides were examined in aqueous solution and in the presence of membrane models (lipid monolayers, micelles, lipid bilayers and vesicles). A combined approach of bioinformatics analyses, biological activity assays, surface pressure measurements, synchrotron radiation circular dichroism spectroscopy, and oriented circular dichroism spectroscopy were employed. Both peptides were able to adsorb at a lipid-air interface with a negative surface charge density, and efficiently disturb the lipid surface packing. A disorder-to-helix transition was observed on the secondary structure of both peptides when either in a non-polar environment or interacting with model membranes containing a negative net charge density. The binding of both temporin-Ra and temporin-Rb to membrane models is modulated by the presence of negatively charged lipids in the membrane. The amphipathic helix induced in temporin-Ra is oriented parallel to the membrane surface in negatively charged or in zwitterionic lipid bilayers, with no tendency for realignment after binding. Temporin-Rb, instead, assumes a ß-sheet conformation when deposited into oriented stacked lipid bilayers. Due to their short size and simple composition, both peptides are quite attractive for the development of new classes of peptide-based anti-infective drugs.

Effect of setting data collection parameters on the reliability of a circular dichroism spectrum.

Circular dichroism (CD) spectroscopy is a well-established biophysical technique used to investigate the structure of molecules. The analysis of a protein CD spectrum depends on the quality of the original CD data, which can be affected by the sample purity, background absorption of the additives/solvent/buffer, the choice of the parameters used for data collection, etc. In this paper, the CD spectrum of myoglobin was used as a model to exploit how variations on each data collection parameter could affect the final protein CD spectrum and, the subsequent effect of them on the quantitative analysis of protein secondary structure. Bioinformatics analysis carried out with SESCA package and PDBMD2CD server predicted a theoretical myoglobin CD spectrum, and a Monte Carlo-like model was implemented to estimate the uncertainty in secondary structure predictions performed with CDSSTR, Selcon 3 and ContinLL algorithms. An inappropriate choice of data collection parameters can lead to a misinterpretation of the CD data in terms of the protein structural content.

Anticandidal activity of synthetic peptides: Mechanism of action revealed by scanning electron and fluorescence microscopies and synergism effect with nystatin.

Candida albicans has emerged as a major public health problem in recent decades. The most important contributing factor is the rapid increase in resistance to conventional drugs worldwide. Synthetic antimicrobial peptides (SAMPs) have attracted substantial attention as alternatives and/or adjuvants in therapeutic treatments due to their strong activity at low concentrations without apparent toxicity. Here, two SAMPs, named Mo-CBP3 -PepI (CPAIQRCC) and Mo-CBP3 -PepII (NIQPPCRCC), are described, bioinspired by Mo-CBP3 , which is an antifungal chitin-binding protein from Moringa oleifera seeds. Furthermore, the mechanism of anticandidal activity was evaluated as well as their synergistic effects with nystatin. Both peptides induced the production of reactive oxygen species (ROS), cell wall degradation, and large pores in the C. albicans cell membrane. In addition, the peptides exhibited high potential as adjuvants because of their synergistic effects, by increasing almost 50-fold the anticandidal activity of the conventional antifungal drug nystatin. These peptides have excellent potential as new drugs and/or adjuvants to conventional drugs for treatment of clinical infections caused by C. albicans.

Unveiling the binding and orientation of the antimicrobial peptide Plantaricin 149 in zwitterionic and negatively charged membranes.

Antimicrobial peptides are a large group of natural compounds which present promising properties for the pharmaceutical and food industries, such as broad-spectrum activity, potential for use as natural preservatives, and reduced propensity for development of bacterial resistance. Plantaricin 149 (Pln149), isolated from Lactobacillus plantarum NRIC 149, is an intrinsically disordered peptide with the ability to inhibit bacteria from the Listeria and Staphylococcus genera, and which is capable of promoting inhibition and disruption of yeast cells. In this study, the interactions of Pln149 with model membranes composed of zwitterionic and/or anionic phospholipids were investigated using a range of biophysical techniques, including isothermal titration calorimetry, surface tension measurements, synchrotron radiation circular dichroism spectroscopy, oriented circular dichroism spectroscopy, and optical microscopy, to elucidate these peptides' mode of interactions and provide insight into their functional roles. In anionic model membranes, the binding of Pln149 to lipid bilayers is an endothermic process and induces a helical secondary structure in the peptide. The helices bind parallel to the surfaces of lipid bilayers and can promote vesicle disruption, depending on peptide concentration. Although Pln149 has relatively low affinity for zwitterionic liposomes, it is able to adsorb at their lipid interfaces, disturbing the lipid packing, assuming a similar parallel helix structure with a surface-bound orientation, and promoting an increase in the membrane surface area. Such findings can explain the intriguing inhibitory action of Pln149 in yeast cells whose cell membranes have a significant zwitterionic lipid composition.

Characterization of esterase activity from an Acetomicrobium hydrogeniformans enzyme with high structural stability in extreme conditions.

The biotechnological and industrial uses of thermostable and organic solvent-tolerant enzymes are extensive and the investigation of such enzymes from microbiota present in oil reservoirs is a promising approach. Searching sequence databases for esterases from such microbiota, we have identified in silico a potentially secreted esterase from Acetomicrobium hydrogeniformans, named AhEst. The recombinant enzyme was produced in E. coli to be used in biochemical and biophysical characterization studies. AhEst presented hydrolytic activity on short-acyl-chain p-nitrophenyl ester substrates. AhEst activity was high and stable in temperatures up to 75 °C. Interestingly, high salt concentration induced a significant increase of catalytic activity. AhEst still retained ~ 50% of its activity in 30% concentration of several organic solvents. Synchrotron radiation circular dichroism and fluorescence spectroscopies confirmed that AhEst displays high structural stability in extreme conditions of temperature, salinity, and organic solvents. The enzyme is a good emulsifier agent and is able to partially reverse the wettability of an oil-wet carbonate substrate, making it of potential interest for use in enhanced oil recovery. All the traits observed in AhEst make it an interesting candidate for many industrial applications, such as those in which a significant hydrolytic activity at high temperatures is required.

Interaction of an esophageal MEG protein from schistosomes with a human S100 protein involved in inflammatory response.

BACKGROUND: The Micro-Exon Gene-14 (MEG-14) displays a remarkable structure that allows the generation of antigenic variation in Schistosomes. Previous studies showed that the soluble portion of the MEG-14 protein displays features of an intrinsically disordered protein and is expressed exclusively in the parasite esophageal gland. These features indicated a potential for interaction with host proteins present in the plasma and cells from ingested blood. METHODS: A yeast two-hybrid experiment using as bait the soluble domain of Schistosoma mansoni MEG-14 (sMEG-14) against a human leukocyte cDNA library was performed. Pull-down and surface plasmon resonance (SPR) experiments were used to validate the interaction between sMEG-14 and human S100A9. Synchrotron radiation circular dichroism (SRCD) were used to detect structural changes upon interaction between sMEG-14 and human S100A9. Feeding of live parasites with S100A9 attached to a fluorophore allowed the tracking of the fate of this protein in the parasite digestive system. RESULTS: S100A9 interacted with sMEG-14 consistently in yeast two-hybrid assay, pull-down and SPR experiments. SRCD suggested that MEG-14 acquired a more regular structure as a result of the interaction with S100A9. Accumulation of recombinant S100A9 in the parasite's esophageal gland, when ingested by live worms suggests that such interaction may occur in vivo. CONCLUSION: S100A9, a protein previously described to be involved in modulation of inflammatory response, was found to interact with sMEG-14. GENERAL SIGNIFICANCE: Our results allow proposing a mechanism involving MEG-14 for the parasite to block inflammatory signaling, which would occur upon release of S100A9 when ingested blood cells are lysed.

Advantages of synchrotron radiation circular dichroism spectroscopy to study intrinsically disordered proteins.

The unordered secondary structural content of an intrinsically disordered protein (IDP) is susceptible to conformational changes induced by many different external factors, such as the presence of organic solvents, removal of water, changes in temperature, binding to partner molecules, and interaction with lipids and/or other ligands. In order to characterize the high-flexibility nature of an IDP, circular dichroism (CD) spectroscopy is a particularly useful method due to its capability of monitoring both subtle and remarkable changes in different environments, relative ease in obtaining measurements, the small amount of sample required, and the capability for sample recovery (sample not damaged) and others. Using synchrotron radiation as the light source for CD spectroscopy represents the state-of-the-art version of this technique with feasibility of accessing the lower wavelength UV region, and therefore presenting a series of advantages over conventional circular dichroism (cCD) to monitor a protein conformational behavior, check protein stability, detect ligand binding, and many others. In this paper, we have performed a comparative study using cCD and SRCD methods for investigating the secondary structure and the conformational behavior of natively unfolded proteins: MEG-14 and soybean trypsin inhibitor. We show that the SRCD technique greatly improves the analysis and accuracy of the studies on the conformations of IDPs.

Deconstructing the DGAT1 enzyme: Binding sites and substrate interactions.

Diacylglycerol acyltransferase 1 (DGAT1) is a microsomal membrane enzyme responsible for the final step in the synthesis of triacylglycerides. Although DGATs from a wide range of organisms have nearly identical sequences, there is little structural information available for these enzymes. The substrate binding sites of DGAT1 are predicted to be in its large luminal extramembranous loop and to include common motifs with acyl-CoA cholesterol acyltransferase enzymes and the diacylglycerol binding domain found in protein kinases. In this study, synthetic peptides corresponding to the predicted binding sites of DGAT1 enzyme were examined using synchrotron radiation circular dichroism spectroscopy, fluorescence emission and adsorption onto lipid monolayers to determine their interactions with substrates associated with triacylglyceride synthesis (oleoyl-CoA and dioleoylglycerol). One of the peptides, Sit1, which includes the FYxDWWN motif common to both DGAT1 and acyl-CoA cholesterol acyltransferase, changes its conformation in the presence of both substrates, suggesting its capability to bind their acyl chains. The other peptide (Sit2), which includes the putative diacylglycerol binding domain HKWCIRHFYKP found in protein kinase C and diacylglycerol kinases, appears to interact with the charged headgroup region of the substrates. Moreover, in an extended-peptide which contains Sit1 and Sit2 sequences separated by a flexible linker, larger conformational changes were induced by both substrates, suggesting that the two binding sites may bring the substrates into close proximity within the membrane, thus catalyzing the formation of the triacylglyceride product.

Novel lipid-interaction motifs within the C-terminal domain of Septin10 from Schistosoma mansoni.

Septins are cytoskeletal proteins and their interaction with membranes is crucial for their role in various cellular processes. Septins have polybasic regions (PB1 and PB2) which are important for lipid interaction. Earlier, we and others have highlighted the role of the septin C-terminal domain (CTD) to membrane interaction. However, detailed information on residues/group of residues important for such feature is lacking. In this study, we investigate the lipid-binding profile of Schistosoma mansoni Septin10 (SmSEPT10) using PIP strip and Langmuir monolayer adsorption assays. Our findings highlight the CTD as the primary domain responsible for lipid interaction in SmSEPT10, showing binding to phosphatidylinositol phosphates. SmSEPT10 CTD contains a conserved polybasic region (PB3) present in both animals and fungi septins, and a Lys (K367) within its putative amphipathic helix (AH) that we demonstrate as important for lipid binding. PB3 deletion or mutation of this Lys (K367A) strongly impairs lipid interaction. Remarkably, we observe that the AH within a construct lacking the final 43 amino acid residues is insufficient for lipid binding. Furthermore, we investigate the homocomplex formed by SmSEPT10 CTD in solution by cross-linking experiments, CD spectroscopy, SEC-MALS and SEC-SAXS. Taken together, our studies define the lipid-binding region in SmSEPT10 and offer insights into the molecular basis of septin-membrane binding. This information is particularly relevant for less-studied non-human septins, such as SmSEPT10.

Ordered mesoporous silicas for potential applications in solid vaccine formulations.

In an effort to develop efficient vaccine formulations, the use of ordered mesoporous silica (SBA-15) as an antigen carrier has been investigated. SBA-15 has required properties such as high surface area and pore volume, including narrow pore size distribution to protect antigens inside its matrix. This study aimed to examine the impact of solvent removal methods, specifically freeze-drying and evaporation on the intrinsic properties of an immunogenic complex. The immunogenic complexes, synthesized and incorporated with BSA, were characterized by various physicochemical techniques. Small Angle X-ray Scattering measurements revealed the characteristic reflections associated to pure SBA-15, indicating the preservation of the silica mesostructured following BSA incorporation and the formation of BSA aggregates within the macropore region. Nitrogen Adsorption Isotherm measurements demonstrated a decrease in surface area and pore volume for all samples, indicating that the BSA was incorporated into the SBA-15 matrix. Fluorescence spectroscopy evidenced that the tryptophan residues in BSA inside SBA-15 or in solution displayed similar spectra, showing the preservation of the aromatic residues' environment. The Circular Dichroism spectra of BSA in both conditions suggest the preservation of its native secondary structure after the encapsulation process. The immunogenic analysis with the detection of anti-BSA IgG did not give any significant difference between the non-dried, freeze-dried or evaporated groups. However, all groups containing BSA and SBA-15 showed results almost three times higher than the groups with pure BSA (control group). These facts indicate that none of the BSA incorporation methods interfered with the immunogenicity of the complex. In particular, the freeze-dried process is regularly used in the pharmaceutical industry, therefore its adequacy to produce immunogenic complexes was proved Furthermore, the results showed that SBA-15 increased the immunogenic activity of BSA.

Contribution of the Tyr-1 in Plantaricin149a to disrupt phospholipid model membranes.

Plantaricin149a (Pln149a) is a cationic antimicrobial peptide, which was suggested to cause membrane destabilization via the carpet mechanism. The mode of action proposed to this antimicrobial peptide describes the induction of an amphipathic α-helix from Ala7 to Lys20, while the N-terminus residues remain in a coil conformation after binding. To better investigate this assumption, the purpose of this study was to determine the contributions of the Tyr1 in Pln149a in the binding to model membranes to promote its destabilization. The Tyr to Ser substitution increased the dissociation constant (KD) of the antimicrobial peptide from the liposomes (approximately three-fold higher), and decreased the enthalpy of binding to anionic vesicles from -17.2 kcal/mol to -10.2 kcal/mol. The peptide adsorption/incorporation into the negatively charged lipid vesicles was less effective with the Tyr1 substitution and peptide Pln149a perturbed the liposome integrity more than the analog, Pln149S. Taken together, the peptide-lipid interactions that govern the Pln149a antimicrobial activity are found not only in the amphipathic helix, but also in the N-terminus residues, which take part in enthalpic contributions due to the allocation at a lipid-aqueous interface.

Interactions of amphipathic α-helical MEG proteins from Schistosomamansoni with membranes.

Micro Exon Gene (MEG) proteins are thought to play major roles in the infection and survival of parasitic Schistosoma mansoni worms in host organisms. Here, the physical chemical properties of two small MEG proteins found in the genome of S. mansoni, named MEG-24 and MEG-27, were examined by a combination of biophysical techniques such as differential scanning calorimetry, tensiometry, circular dichroism, fluorescence, and electron spin resonance spectroscopies. The proteins are surface active and structurally arranged as cationic amphipathic α-helices that can associate with lipid membranes and cause their disruption. Upon adsorption to lipid membranes, MEG-27 strongly affects the fluidity of erythrocyte ghost membranes, whereas MEG-24 forms pores in erythrocytes without modifying the ghost membrane fluidity. Whole-mount in situ hybridization experiments indicates that MEG-27 and MEG-24 transcripts are located in the parasite esophagus and subtegumental cells, respectively, suggesting a relevant role of these proteins in the host-parasite interface. Taken together, these characteristics lead us to propose that these MEG proteins may interact with host cell membranes and potentially modulate the immune process using a similar mechanism as that described for α-helical membrane-active peptides.

RcAlb-PepII, a synthetic small peptide bioinspired in the 2S albumin from the seed cake of Ricinus communis, is a potent antimicrobial agent against Klebsiella pneumoniae and Candida parapsilosis.

Antimicrobial peptides (AMPs) are important constituents of the innate immunity system of all living organisms. They participate in the first line of defense against invading pathogens such as viruses, bacteria, and fungi. In view of the increasing difficulties to treat infectious diseases due to the emergence of antibiotic-resistant bacterial strains, AMPs have great potential to control infectious diseases in humans and animals. In this study, two small peptides, RcAlb-PepI and RcAlb-PepII, were designed based on the primary structure of Rc-2S-Alb, a 2S albumin from the seed cake of Ricinus communis, and their antimicrobial activity assessed. RcAlb-PepII strongly inhibited the growth of Klebsiella pneumoniae and Candida parapsilosis, and induced morphological alterations in their cell surface. C. parapsilosis exposed to RcAlb-PepII presented higher cell membrane permeabilization and elevated content of reactive oxygen species. RcAlb-PepII also degraded and reduced the biofilm formation in C. parapsilosis and in K. pneumonia cells. Experimentally, RcAlb-PepII was not hemolytic and had low toxicity to mammalian cells. These are advantageous characteristics, which suggest that RcAlb-PepII is safe and apparently effective for its intended use and has great potential for the future development of an antimicrobial agent with the ability to kill or inhibit K. pneumoniae and C. parapsilosis cells.

Synthetic antimicrobial peptides: From choice of the best sequences to action mechanisms.

The emergence of antibiotic-resistant microbes has stimulated research worldwide seeking new biologically active molecules. In this respect, synthetic antimicrobial peptides (SAMPs) have been suggested to overcome this problem. Although there are some online servers that provide putative SAMPs from protein sequences, the choice of the best peptide sequences for further analysis is still difficult. Therefore, the goal of this paper is not to launch a new tool but to provide a friendly workflow to characterize and predict potential SAMPs by employing existing tools. Using this proposed workflow, two peptides (PepGAT and PepKAA) were obtained and extensively characterized. These peptides damaged microbial membranes and cell walls, and induced overproduction of reactive oxygen species (ROS). Both peptides were found to assume random coil secondary structure in aqueous solution, organic solvent, and upon binding to negatively charged lipid systems. Peptides were also able to degrade formed biofilms but not to prevent biofilm formation. PepGAT was not resistant to proteolysis, whereas PepKAA was resistant to pepsin but not to pancreatin. Furthermore, both presented no hemolytic activity against red blood cells, even at a 10-fold higher concentration than the antimicrobial concentration. The pipeline proposed here is an easy way to design new SAMPs for application as alternatives to develop new drugs against human pathogenic microorganisms.

The GRASP domain in golgi reassembly and stacking proteins: differences and similarities between lower and higher Eukaryotes.

The Golgi complex is part of the endomembrane system and is responsible for receiving transport cargos from the endoplasmic reticulum and for sorting and targeting them to their final destination. To perform its function in higher eukaryotic cells, the Golgi needs to be correctly assembled as a flattened membrane sandwich kept together by a protein matrix. The precise mechanism controlling the Golgi cisternae assembly is not yet known, but it is widely accepted that the Golgi Reassembly and Stacking Protein (GRASP) is a main component of the Golgi protein matrix. Unlike mammalian cells, which have two GRASP genes, lower eukaryotes present only one gene and distinct Golgi cisternae assembly. In this study, we performed a set of biophysical studies to get insights on the structural properties of the GRASP domains (DGRASPs) from both human GRASP55 and GRASP65 and compare them with GRASP domains from lower eukaryotes (Saccharomyces cerevisiae and Cryptococcus neoformans). Our data suggest that both human DGRASPs are essentially different from each other and that DGRASP65 is more similar to the subgroup of DGRASPs from lower eukaryotes in terms of its biophysical properties. GRASP55 is present mainly in the Golgi medial and trans faces, which are absent in both fungi, while GRASP65 is located in the cis-Golgi. We suggest that the GRASP65 gene is more ancient and that its paralogue GRASP55 might have appeared later in evolution, together with the medial and trans Golgi faces in mammalians.

Structural and enzymatic characterization of Peruvianin­I, the first germin-like protein with proteolytic activity.

The germin-like protein (GLP) purified from Thevetia peruviana, Peruvianin-I, is the only one described as having proteolytic activity. Therefore, the goal of this study was to investigate the structural features responsible for its enzymatic activity. Although the amino acid sequence of Peruvianin-I showed high identity with other GLPs, it exhibited punctual mutations, which were responsible for the absence of oxalate oxidase activity. The phylogenetic analysis showed that Peruvianin-I does not belong to any classification of GLP subfamilies. Moreover, Peruvianin-I contains a catalytic triad found in all plant cysteine peptidases. Molecular docking simulations confirmed the role of the catalytic triad in its proteolytic activity. Synchrotron radiation circular dichroism assays confirmed that Peruvianin-I was stable at pH ranging from 5.0 to 8.0 and that it presented significant structural changes only above 60 °C. The addition of iodoacetamide caused changes in its native conformation, but only a slight effect was observed after adding a reducing agent. This study reports an unusual protein with germin-like structure, lacking typical oxalate oxidase activity. Instead, the proteolytic activity observed suggests that the protein is a cysteine peptidase. These structural peculiarities make Peruvianin­I an interesting model for further understanding of the action of laticifer fluids in plant defense.

Structural characterization of novel chitin-binding lectins from the genus Artocarpus and their antifungal activity.

Two novel chitin-binding lectins from seeds of Artocarpus genus were described in this paper, one from A. integrifolia (jackfruit) and one from A. incisa (breadfruit). They were purified from saline crude extract of seeds using affinity chromatography on chitin column, size-exclusion chromatography and reverse-phase chromatography on the C-18 column. Both are 14 kDa proteins, made up of 3 chains linked by disulfide bonds. The partial amino acid sequences of the two lectins showed they are homologous to each other but not to other plant chitin-binding proteins. Thus, they cannot be classified in any known plant chitin-binding protein family, particularly because of their inter-chain covalent bonds. Their circular dichroism spectra and deconvolution showed a secondary structure content of beta-sheet and unordered elements. The lectins were thermally stable until 80 degrees C and structural changes were observed below pH 6. Both lectins inhibited the growth of Fusarium moniliforme and Saccharomyces cerevisiae, and presented hemagglutination activity against human and rabbit erythrocytes. These lectins were denoted jackin (from jackfruit) and frutackin (from breadfruit).

Calliandra selloi Macbride trypsin inhibitor: isolation, characterization, stability, spectroscopic analyses.

A trypsin inhibitor was purified from Calliandra selloi Macbride seeds (CSTI). SDS-PAGE under non-reducing conditions showed a single band of approximately 20,000 Da, while under reducing conditions two bands of 16,000 and 6000 Da were observed, indicating that CSTI consists of two polypeptide chains. Molecular masses of 20,078 and 20,279 were obtained by mass spectrometry, although only one pI of 4.0 was observed and one peak was obtained by reversed phase chromatography. Amino-terminal sequence analysis showed homology to Kunitz-type inhibitors. CSTI was able to inhibit trypsin (Ki 2.21 x 10(-7)M), alpha-chymotrypsin (Ki 4.95 x 10(-7)M) and kallikrein (Ki 4.20 x 10(-7)M) but had no effect on elastase. Trypsin inhibitory activity was stable over a wide range of pH and temperature. CSTI was particularly susceptible to DTT treatment, followed by addition of iodoacetamide. Far-UV circular dichroism measurements revealed that CSTI is a beta-II protein. Thermal unfolding showed a two-state transition with a midpoint at 68 degrees C. Far-UV CD spectra of CSTI at pH extremes showed little changes, while more pronounced differences in near-UV CD spectra were detected. Remarkably, treatment with 1mM DTT caused very slight changes in the far-UV CD spectrum, and only after carbamidomethylation was there was a marked loss observed in secondary structure.

Going deep into protein secondary structure with synchrotron radiation circular dichroism spectroscopy.

Circular dichroism (CD) spectroscopy is a fast, powerful, well-established, and widely used analytical technique in the biophysical and structural biology community to study protein secondary structure and to track changes in protein conformation in different environments. The use of the intense light of a synchrotron beam as the light source for collecting CD measurements has emerged as an enhanced method, known as synchrotron radiation circular dichroism (SRCD) spectroscopy, that has several advantages over the conventional CD method, including a significant spectral range extension for data collection, deeper access to the lower limit (cut-off) of conventional CD spectroscopy, an improved signal-to-noise ratio to increase accuracy in the measurements, and the possibility to collect measurements in highly absorbing solutions. In this review, we discuss different applications of the SRCD technique by researchers from Latin America. In this context, we specifically look at the use of this method for examining the secondary structure and conformational behavior of proteins belonging to the four main classes of the hierarchical protein domain classification CATH (Class, Architecture, Topology, Homology) database, focusing on the advantages and improvements associated with SRCD spectroscopy in terms of characterizing proteins composed of different structural elements.

Environmental Factors Modulating the Stability and Enzymatic Activity of the Petrotoga mobilis Esterase (PmEst).

Enzymes isolated from thermophilic organisms found in oil reservoirs can find applications in many fields, including the oleochemical, pharmaceutical, bioenergy, and food/dairy industries. In this study, in silico identification and recombinant production of an esterase from the extremophile bacteria Petrotoga mobilis (designated PmEst) were performed. Then biochemical, bioinformatics and structural characterizations were undertaken using a combination of synchrotron radiation circular dichroism (SRCD) and fluorescence spectroscopies to correlate PmEst stability and hydrolytic activity on different substrates. The enzyme presented a high Michaelis-Menten constant (KM 0.16 mM) and optimum activity at ~55°C for p-nitrophenyl butyrate. The secondary structure of PmEst was preserved at acid pH, but not under alkaline conditions. PmEst was unfolded at high concentrations of urea or guanidine through apparently different mechanisms. The esterase activity of PmEst was preserved in the presence of ethanol or propanol and its melting temperature increased ~8°C in the presence of these organic solvents. PmEst is a mesophilic esterase with substrate preference towards short-to medium-length acyl chains. The SRCD data of PmEst is in agreement with the prediction of an α/ß protein, which leads us to assume that it displays a typical fold of esterases from this family. The increased enzyme stability in organic solvents may enable novel applications for its use in synthetic biology. Taken together, our results demonstrate features of the PmEst enzyme that indicate it may be suitable for applications in industrial processes, particularly, when the use of polar organic solvents is required.