Random Coil Secondary Structure Research Articles

Self-assembly and hydrogelation of a potential bioactive peptide derived from quinoa proteins

In this work the identification of peptides derived from quinoa proteins which could potentially self-assemble, and form hydrogels was carried out with TANGO, a statistical mechanical based algorithm that predicts β-aggregate propensity of peptides. Peptides with the highest aggregate propensity were subjected to gelling screening experiments from which the most promising bioactive peptide with sequence KIVLDSDDPLFGGF was selected. The self-assembling and hydrogelation properties of the C-terminal amidated peptide (KIVLDSDDPLFGGF-NH2) were studied. The effect of concentration, pH, and temperature on the secondary structure of the peptide were probed by circular dichroism (CD), while its nanostructure was studied by transmission electron microscopy (TEM) and small-angle neutron scattering (SANS). Results revealed the existence of random coil, α-helix, twisted β-sheet, and well-defined β-sheet secondary structures, with a range of nanostructures including elongated fibrils and bundles, whose proportion was dependant on the peptide concentration, pH, or temperature. The self-assembly of the peptide is demonstrated to follow established models of amyloid formation, which describe the unfolded peptide transiting from an α-helix-containing intermediate into β-sheet-rich protofibrils. The self-assembly is promoted at high concentrations, elevated temperatures, and pH values close to the peptide isoelectric point, and presumably mediated by hydrogen bond, hydrophobic and electrostatic interactions, and π-π interactions (from the F residue). At 15 mg/mL and pH 3.5, the peptide self-assembled and formed a self-supporting hydrogel exhibiting viscoelastic behaviour with G' (1 Hz) ~2300 Pa as determined by oscillatory rheology measurements. The study describes a straightforward method to monitor the self-assembly of plant protein derived peptides; further studies are needed to demonstrate the potential application of the formed hydrogels in food and biomedicine.

Physicochemical, functional, and antioxidant properties of black soldier fly larvae protein.

This study explores the multifaceted attributes of black soldier fly larvae protein (BSFLP), focusing on its physicochemical, functional, and antioxidant properties. BSFLP is characterized by 16 amino acids, with a predominant random coil secondary structure revealed by circular dichroism spectra. Differential scanning calorimetry indicates a substantial thermal denaturation temperature of 97.63°C. The protein exhibits commendable solubility, emulsification, and foaming properties in alkaline and low-salt environments, albeit with reduced water-holding capacity and foam stability under elevated alkaline and high-temperature conditions. In vitro assessments demonstrate that BSFLP displays robust scavenging proficiency against 2,2-diphenyl-1-picrylhydrazyl, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), and hydroxyl radicals, with calculated EC50 values of 1.90±0.57, 0.55±0.01, and 1.14±0.02mg/mL, respectively, along with notable reducing capabilities. Results from in vivo antioxidant experiments reveal that BSFLP, administered at doses of 300 and 500mg/kg, significantly enhances the activities of antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase) (p<0.05) while simultaneously reducing malondialdehyde levels in both serum and tissues of d-galactose-induced oxidative stress in mice. Moreover, the protein effectively attenuates oxidative damage in liver and hippocampal tissues. These findings underscore the potential utility of BSFLP as a natural antioxidant source, with applications spanning the food, pharmaceutical, and cosmetic industries. PRACTICAL APPLICATION: Black soldier fly larvae protein emerges as an environmentally sustainable reservoir of natural antioxidants, holding significant promise for the food, pharmaceutical, and cosmetic sectors. Its advantageous amino acid composition, robust thermal resilience, and impressive functional attributes position it as a compelling subject for continued investigation and advancement in various applications.

Endotoxin accelerates insulin amyloid formation and inactivates insulin signal transduction

Aims and objectivesThe aim of this study is to discuss the influence of endotoxin on insulin amyloid formation, to provide guidance for therapeutic insulin preparation and storage. Materials and methodsThe ThT and ANS binding assays were applied to characterize the dynamics curve of insulin amyloid formation with the presence or absence of endotoxin. The morphological structures of intermediate and mature insulin fibrils were observed with SEM and TEM. Secondary structural changes of insulin during fibriliation were examined with CD, FTIR and Raman spectral analysis. The cytotoxic effects of oligomeric and amyloidogenic insulin aggregates were detected using a cck-8 cell viability assay kit. The influence of endotoxin on insulin efficacy was analyzed by monitoring the activation of insulin signal transduction. Key findingsThT analysis showed that endotoxin, regardless of species, accelerated insulin fibrils formation in a dose-dependent manner, as observed with a shorter lag phase. ANS binding assay demonstrated endotoxin provoked the exposure of insulin hydrophobic patches. The results of SEM and TEM data displayed that endotoxin drove insulin to cluster into dense and viscous form, with thicker and stronger filaments. Based on CD, FTIR and Raman spectra, endotoxin promoted the transition of α-helix to random coil and β-strand secondary structures during insulin aggregation. Insulins in both oligomeric and amyloidogenic forms were cytotoxic to HepG2 cells, with the former being more severe. Finally, the efficacy of endotoxin treated insulin obviously decreased. SignificanceOur studies revealed that endotoxin disrupts the structural integrity of insulin and promotes its amyloidosis. These findings offered theoretical guidance for insulin storage and safe utilization, as well as pointing up a new direction for insulin resistance research.

Novel Peptide from the Hydrolysate of Hybrid Sturgeon (Acipenseridae) Spinal Cord: Isolation, Identification, and Anti-proliferative Effects in Human Cervix Cancer Cells.

Anti-proliferative peptides have recently attracted attention for their excellent bioactivity and biocompatibility. In this paper, five novel anti-proliferative peptides were identified from the hydrolysate of hybrid sturgeon spinal cord (HSSC). In addition, the structure-activity relationship of the novel anti-proliferative peptides was explored. In vitro experiments indicated that the peptide "VDSVLDVVRK" presented the highest inhibition of HeLa cell growth in all samples (IC50 = 2.5 μM). VDSVLDVVRK showed a random coil secondary structure and nanomicelles in the tumor microenvironment. Transmission electron microscopy results confirmed that nanomicelles disassemble as the concentration of VDSVLDVVRK decreases. Furthermore, VDSVLDVVRK could induce HeLa cell apoptosis by increasing the expression of Cyt-c (98.65 ± 1.85%, p < 0.01) and caspase-9 (39.85 ± 1.81%, p < 0.01). In this study, the anti-proliferative mechanism of the HSSC peptide was discussed, which provided a theoretical basis for the research and development of anti-proliferative functional food.

Farnesylation and lipid unsaturation are critical for the membrane binding of the C-terminal segment of G-Protein Receptor Kinase 1

Many proteins are modified by the covalent addition of different types of lipids, such as myristoylation, palmitoylation and prenylation. Lipidation is expected to promote membrane association of proteins. Visual phototransduction involves many lipid-modified proteins. The G-Protein-coupled receptor of rod photoreceptors, rhodopsin, is inactivated by G-Protein-coupled Receptor Kinase 1 (GRK1). The C-terminus of GRK1 is farnesylated and its truncation has been shown to result in a very high decrease of its enzymatic activity, most likely because of the loss of its membrane localization. Little information is available on the membrane binding of GRK1 as well as of most prenylated proteins. Measurements of the membrane binding of the non-farnesylated and farnesylated C-terminal segment of GRK1 were thus performed using lipids typical of those found in rod outer segment disk membranes. Their random coil secondary structure was determined using circular dichroism and infrared spectroscopy. The non-farnesylated C-terminal segment of GRK1 has no surface activity. In contrast, the farnesylated C-terminal segment of GRK1 shows a particularly strong binding to lipid monolayers bearing at least one unsaturated fatty acyl chain. No binding is observed in the presence of monolayers of saturated phospholipids, in agreement with the low affinity of farnesylated Ras proteins for lipids in the liquid-ordered state. Altogether, these data demonstrate that the farnesyl group of the C-terminal segment of GRK1 is mandatory for its membrane binding, which is favored by particular lipids or lipid mixtures. This information will also be useful for the understanding of the membrane binding of other prenylated proteins.

The Effect of Enzymatic-TGase Cross-Linking on the Structure of Soy Protein Isolate Based on Multiplex Spectroscopy

The functional properties of soy protein isolate (SPI) after alkaline protease digestion and transglutaminase (TGase) catalyzed polymerization is correlated with its internal molecule structure. The structure of SPI modified by alkaline protease digestion-TGase by using ultraviolet (UV) and Fourier transform infrared (FT-IR) and circular dichroism spectroscopy were characterized. The UV spectroscopy analysis showed that the cross-linking of SPI by alkaline protease digestion-TGase induced the reorganization and polymerization of small polypeptide chains, and the absorption intensity of Tyr and Trp increased, which demonstrated that the side chain structure of the SPI polypeptide chains modified by TGase was changed. The results of FT-IR showed that the absorption peaks of amide I and II segments shifted after TGase cross-linked SPI, and part of beta-sheet secondary structures was transformed into alpha-helix, indicated that the intramolecular hydrogen bonds of the modified SPI were changed. By circular dichroism spectroscopy showed that the content of random coil and alpha-helix secondary structure of SPI modified by TGase increased and the content of beta-sheet structure decreased.

Tip-enhanced Raman spectroscopy of Aβ(1-42) fibrils

Using tip-enhanced Raman spectroscopy (TERS) imaging with a lateral optical resolution of ~16 nm, the heterogeneity of Aβ(1-42) fibrils implicated in Alzheimer’s disease is investigated. The amount and spatial distribution of TERS bands assigned to aromatic amino acid residues (histidine, tyrosine and phenylalanine) and to parallel β-sheet and random coil secondary structures suggest that both the surface and core of Aβ(1-42) fibrils are probed, confirm the twisted nature of the fibrils and reveal the presence of several β-sheet and disordered fibril regions with different hydrophilicity. This study completes and bears out literature TERS data on Aβ(1-42) amyloid fibrils.

PH- and concentration-dependent supramolecular self-assembly of a naturally occurring octapeptide.

Peptide-based biopolymers represent highly promising biocompatible materials with multiple applications, such as tailored drug delivery, tissue engineering and regeneration, and as stimuli-responsive materials. Herein, we report the pH- and concentration-dependent self-assembly and conformational transformation of the newly synthesized octapeptide PEP-1. At pH 7.4, PEP-1 forms β-sheet-rich secondary structures into fractal-like morphologies, as verified by circular dichroism (CD), Fourier-transform infrared (FTIR) spectroscopy, thioflavin T (ThT) fluorescence spectroscopy assay, and atomic force microscopy (AFM). Upon changing the pH value (using pH 5.5 and 13.0), PEP-1 forms different types of secondary structures and resulting morphologies due to electrostatic repulsion between charged amino acids. PEP-1 can also form helical or random-coil secondary structures at a relatively low concentration. The obtained pH-sensitive self-assembly behavior of the target octapeptide is expected to contribute to the development of novel drug nanocarrier assemblies.

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.

Effect of multi-frequency countercurrent ultrasound treatment on extraction optimization, functional and structural properties of protein isolates from Walnut (Juglans regia L.) meal.

This study evaluated the effects of ultrasound treatment on walnut meal protein (WMP) extraction and techno-functional properties. The Box-Behnken Design (BBD) was adopted for the optimization of the traditional and ultrasound-assisted extraction (UAE) processes. Standard protocols were used to assay the techno-functional characteristics. The extraction models' statistical results exhibited adequacy with the least desirability index of 95.8%. The UAE enhanced the WMP extraction yield, purity, and chemical score by 30.15%, 16.27%, and 9.74%, respectively, while reducing the extraction time by 25% over the control. The emulsion and foam stabilities and bulk density increased by 34.5%, 39.8%, and 6.1%, respectively, over the control. The α-helix decreased while β-sheet, β-turns and random coil secondary structure components increased significantly (p<.05) by 95.76%, 101.3%, 105.1%, and 85.7% correspondingly. The dual-frequency combination (20/40kHz/kHz) was the best frequency mode. WMP could serve as a functional additive in manufactured foods as texture and flavor enhancer. PRACTICAL APPLICATIONS: Walnut meal protein (WMP) has a well-balanced amino acid profile and its economic use could be practically increased as a food ingredient by ultrasound-assisted extraction. By this technique, WMP could be employed for the development of enhanced food ingredients rather than being discarded as animal feed. This study showed a positive effect of ultrasonic-assisted alkaline pretreatment on WMP extraction, functionality and structure characteristics. In addition to process improvement, ultrasound is energy efficient and environmentally friendly. Therefore, the applicability of this technique to improve the functionality of plant proteins from industrial by-products to be included in food products is promising.

Interaction and binding mechanism of cyanidin-3-O-glucoside to ovalbumin in varying pH conditions: A spectroscopic and molecular docking study

Egg ovalbumin (OVA) as a prevalent dietary protein and has the potential to serve as a carrier for unstable bioactive compounds, however, understanding their interaction mechanism is the preliminary step. In this work, the interactions between cyanidin-3-O-glucoside (C3G) and OVA in both acidic and neutral pH environment were investigated by spectroscopic methods and molecular docking analysis. The results revealed that fluorescence quenching mechanism of OVA-C3G was predominantly static. The main acting forces were hydrogen bonds and van der Waals forces under varying pH conditions. However, the binding affinity of C3G to OVA was higher in neutral environment than that in acidic condition. The binding of C3G slightly increased the diameter of the complex, resulting in increase of α-helix, decrease of β-turn, random coil, and total main secondary structure. Moreover, the thermostability of C3G was significantly improved after OVA addition, suggesting its promising application in functional foods.

Enzymatic analysis, structural study and molecular docking of laccase and catalase from B. subtilis SK1 after textile dye exposure

The textile dye decolorizing efficiency of Bacillus subtilis SK1 against 70 mg/L each of Malachite Green, Methyl Orange, Rubine GFL and Red HE3B was observed as 71.7, 73.6, 74.4 and 82.6%, respectively within 3 h. UV–Vis spectroscopy, GC–MS and HPTLC analysis confirmed mineralization of model dyes into its metabolites. Physico-chemical characterization confirmed acidic and hydrophilic nature of both laccase and catalase enzymes. Both enzymes contain dominant random coiled secondary structure (SOPMA tool) and intracellular location (CELLO_v.2.5), however, laccase alone contains two disulfide bridges (CYS_REC tool). The validation of constructed 3D structure (Modeller 9.19) of laccase and catalase enzymes revealed, RAMPAGE- 96.3 and 95.8% residues in favoured region, ProSA- Z score −8.2 and −9.6, respectively and ERRAT-Overall quality factor > 68. Potential energies −1.328 × 106 kJ/mol and −2.685 × 106 kJ/mol remained constant after 1395 and 1545 steps for laccase and catalase, respectively in energy minimization. Molecular docking results showed interaction of Methyl Orange with laccase (Thr260) and catalase (Lys 48), Rubine GFL with laccase (Thr 262) and catalase (His 176) and Red HE3B with laccase (Asn 264, Thr 418, Gly 321, Thr 262 and Gly 378) and catalase (Gln 258). This study provides dye degrading potential of Bacillus subtilis strain SK1 with structurally different textile dyes and vital role of the polar amino acids of laccase and catalase in these interactions.

A Structural and Functional Role for Disulfide Bonds in a Class II Hydrophobin.

Hydrophobins are multifunctional, highly surface active proteins produced in filamentous fungi and can be identified by eight conserved cysteine residues, which form four disulfide bridges. These proteins can be subdivided into two classes based on their hydropathy profiles, solubility, and structures formed upon interfacial assembly. Here, we probe the structural and functional roles of disulfide bonds for a class II hydrophobin in different interfacial contexts by reducing its disulfides with 1,4-dithiothreitol and blocking the free thiols with iodoacetamide and then examining the protein secondary structure, emulsification capability, hydrophobic surface wetting, and solution self-assembly. Changes in circular dichroism spectra upon reduction and blocking of disulfides are consistent with an increase in the level of random coil secondary structure. Emulsification of octane in water using reduced and unreduced forms of class II hydrophobin showed a substantial loss of emulsification ability without disulfides and stable emulsion formation for hydrophobin with disulfides. Additionally, water contact angle measurements performed on polytetrafluoroethylene treated with solutions of reduced and unreduced hydrophobin showed efficient wetting of the hydrophobic surface for unreduced samples only. Lastly, Förster resonance energy transfer (FRET) was used to assess the role of disulfides in self-assembly in solution, and near complete loss of the FRET signal is consistent with a model in which solution self-assembly does not occur after reduction and blocking of the disulfides. From this, we conclude that, in contrast to class I hydrophobins, the disulfides of this class II hydrophobin are required for protein structural stability, surface activity at both liquid-liquid and solid-liquid interfaces, and solution self-assembly.

Spider prey-wrapping silk is an α-helical coiled-coil/β-sheet hybrid nanofiber.

Solid-State NMR results on 13C-Ala/Ser and 13C-Val enriched Argiope argentata prey-wrapping silk show that native, freshly spun aciniform silk nanofibers are dominated by α-helical (∼50% total) and random-coil (∼35% total) secondary structures, with minor β-sheet nanocrystalline domains (∼15% total). This is the most in-depth study to date characterizing the protein structural conformation of the toughest natural biopolymer: aciniform prey-wrapping silks.

Recombinant-phospholipase A2 production and architecture of inclusion bodies are affected by pH in Escherichia coli

Aggregation of recombinant proteins into inclusion bodies (IBs) is the major drawback of heterologous expression in Escherichia coli. Here, we evaluated the effects of a pH shift after expression induction on recombinant phospholipase A2 production and its aggregation in IBs in E. coli Origami™, as compared to cultures with pH maintained at 7.5 or uncontrolled pH. Cultures shifted from 7.5 to pH 6.5 or 8.5 produced ∼15–25% less biomass as compared with those kept at 7.5 or without pH control. The cultures shifted to pH 8.5 showed a ∼50% higher yield of acetate per biomass, and the rPLA2 yield was improved 2.4-fold. Purified IBs formed at pH 8.5 containing ∼50% of rPLA2, were more susceptible to proteinase-K cleavage and bound less thioflavin-T, indicating lower amyloid content, with the concomitant enrichment of α-helical and random-coil secondary structures, as demonstrated by FTIR. Moreover, only one IB per cell was formed at pH 8.5; instead, more than two were observed under the other culture pH conditions. Nevertheless, under uncontrolled pH conditions, ∼300nm larger IBs were observed. Our work presents evidence of the usefulness of recombinant protein expression cultivated at pH 8.5 allowing the reduction of amyloid content in IBs.

Stabilization of a chimeric malaria antigen in separation and purification through efficient inhibition of protease activity by imidazole

Abstract A chimeric antigen M.RCAg-1 of Plasmodium falciparum expressed in Escherichia coli was previously demonstrated inhibiting the growth of malaria parasites in vitro, but its further development has been retarded by the antigen’s instability during the downstream process. In this study, it was definitely demonstrated the instability was caused by the susceptibility of M.RCAg-1 to metalloprotease(s) released from the disintegrated host cells. Interestingly, imidazole showed better inhibition effects on the degradation than EDTA. Hence, a purification procedure was successfully developed to produce M.RCAg-1 with a purity of up to 95% and an overall recovery of nearly 600 mg/L culture. When performed this protocol following the Good Manufacturing Practice regulations, the endotoxin level, the host protein content and residual DNA level, all met the FDA standards. MALDI-TOF MS demonstrated a consistent molecular weight with the theoretical value and CD revealed a mainly disordered random coil secondary structure. Immunizing mice with M.RCAg-1 with Freund’s adjuvant elicited high levels of specific antibodies. Moreover, M.RCAg-1 itself could be stable at 4 °C for up to 6 months. Our results would provide an efficient and robust protocol for the large-scale production of M.RCAg-1 which would warrant the further development of this promising malaria vaccine candidate.

Topochemistry, optical anisotropy and FT-IR microspectroscopy of the cocoon of Lithurgus chrysurus (Hymenoptera, Megachilidae)

A previous study has not revealed the participation of a mucous component in the cocoon wall of the solitary bee, Lithurgus chrysurus, differing from the cocoon structure reported for many other bee species. However, uncertainty remains, because only the median and rear zones of this cocoon type have thus far been analyzed. Here, we studied the front zone of this cocoon, searching its components and their organization, to fill this knowledge gap. Topochemical assays, polarization microscopy and Fourier transform-infrared (FT-IR) microspectroscopy were used to study cross sections from L. chrysurus cocoon. Three main layers differing in structural organization were found to compose the cocoon wall. Silk fibroins were assumed to constitute the filamentous threads of the inner and outer layers and the laminar structure of the intermediate layer. Deduced from its topochemical properties and FT-IR spectral signature, a foamy material containing mucin glycoproteins and carboxylated acid glycosaminoglycans was found in the intermediate layer. FT-IR analysis using a Savitzky-Golay 2nd-derivative and absence of linear dichroism and birefringence phenomena suggest that a random-coil secondary structure predominates in the foam component. Co-existence of α-helical and β-sheet conformations is also hypothesized for the fibroin component of this cocoon. It is thus concluded that in addition to fibroin elements, a mucous component, likely contributed by a Malpighian tubule secretion, integrates the composition of the front zone of the cocoon wall of L. chrysurus. In addition, the FT-IR analysis of the inner layer silk of this cocoon suggests significant differences in comparison to the silk fibroins of the silkworm, and some minor spectral differences in comparison to published data on the honeybee silk, with respect to protein secondary structure.

Effects of konjac glucomannan on heat-induced changes of physicochemical and structural properties of surimi gels

This work demonstrated the protective effects of konjac glucomannan (KGM) on the physicochemical and structural properties of surimi gels subjected to 120°C. The T2 relaxation of LF-NMR which was used to detect water mobility, changed more obviously with the addition of deacetylated KGM than with the native, which indirectly implied that deacetylated KGM more pronouncedly influenced the protein structure. Hydrophobic interaction, ionic, hydrogen, and disulfide bonds, were sheltered by deacetylated KGM to greater extent than by native KGM. Raman spectra showed that the reduction of the main random-coil secondary protein structure at 120°C was more significantly mitigated by deacetylated KGM. The interactions between protein and native or deacetylated KGM were investigated by X-ray diffraction, finding that protein and polysaccharide complexes might be formed. The microstructure of the mixed gels, determined by confocal laser scanning microscopy, demonstrated native and deacetylated KGM helped protein aggregation recover to uniform distribution.

BIOPHYSICAL AND STRUCTURAL ANALYSES OF THE ANTI-ATHEROSCLEROTIC PROPERTIES OF HSP-27

BIOPHYSICAL AND STRUCTURAL ANALYSES OF THE ANTI-ATHEROSCLEROTIC PROPERTIES OF HSP-27

Chemical and thermal influence of the [4Fe–4S]2 + cluster of A/G-specific adenine glycosylase from Corynebacterium pseudotuberculosis

The gram-positive bacteria Corynebacterium pseudotuberculosis, the causative agent of caseous lymphadenitis in livestock significantly reduces productivity and often causes death. The adenine/guanine-specific DNA glycosylase (MutY) prevents mutations in the DNA of the pathogen and a unique feature of the MutY protein family is the [4Fe–4S]2+ cluster that interlinks two protein subdomains. MutY from C. pseudotuberculosis was expressed in E. coli and purified, the CD experiments indicate a high content of α-helices and random coiled secondary structure and a typical near-UV CD fingerprint for the [4Fe–4S]2+ cluster. EDTA and copper sulfate possess a strong destabilizing effect on the [4Fe–4S]2+ cluster. UV–vis and fluorescence spectroscopy results demonstrate that between pH3.0 and 4.0 the integrity of the [4Fe–4S]2+ cluster is destroyed. To investigate the thermal stability of the protein differential scanning calorimetry and fluorescence spectroscopy were used and the Tm was determined to be 45°C. The analysis presented provides information concerning the protein stability under different physio-chemical conditions.