Thermal Denaturation Process Research Articles

Molecular characterization of the effects of Ganoderma Lucidum polysaccharides on the structure and activity of bovine serum albumin.

The investigation about polysaccharides-protein system is attributed to numerous very important applications for pharmaceutical, food, chemical and other industries. In the present work, multi-spectral methods and molecular docking were used to analyze the molecular interactions of polysaccharides from Ganoderma Lucidum (GLP) with bovine serum albumin (BSA). The nonenzymatic glucosylation, fibrillation, thermal stability, and structure information of GLP-BSA system were also studied. The results showed that the formation of GLP-BSA complex by mainly hydrogen-bonding forces resulted in the conformational changes of protein. GLP acted as a stabilizer to increase the thermal stability of BSA solution having a novel and more stable conformational state during the thermal denaturation process. 8-anilino-1-naphthalenesulfonic acid (ANS) fluorescence spectral results suggested that there exist some intermediate state which has low binding ability with ANS in the presence of GLP. The presence of GLP caused a decrease in the formation of beta sheet structures with a lower rate. The fluorescence spectra of BSA glycosylated by GLP confirmed the formation of covalent bonds between BSA and GLP through the Maillard reaction which was also confirmed by using thermogravimetric (TGA) and Fourier transform infrared (FTIR) analysis. In addition, BSA still maintains the esterase-like good activity in the presence of GLP. These results provide a basis for screening the molecular interactions of polysaccharides with protein from the perspective of important food active ingredients.

Binding mechanism of five typical sweeteners with bovine serum albumin

In this work, the interactions between bovine serum albumin (BSA) and five sweeteners including aspartame (APM), acesulfame (AK), sucralose (TGS), sodium cyclamate (SC), and rebaudioside-A (REB-A) have been studied by multispectroscopic techniques, and molecular simulation in order to provide much useful information for the application of new and safer artificial sweeteners. Fluorescence quenching assays indicated that the formation of complexes between sweeteners and BSA mainly induced the fluorescence quenching of protein and the binding site number were about 1 indicting that there is one mainly binding site of APM, AK, TGS, SC, or REB-A in domain of BSA with relatively weak interactions. Molecular modeling results indicated that hydrogen bonding interactions were the mainly binding forces of sweeteners with BSA. Circular dichroism spectra indicated that APM and REB-A obviously induced the secondary structure changes of BSA. The presence of APM increased the fraction of α–Helix of BSA from 65.4% to 73.8%, while the presence of REB-A resulted in decreasing the fraction of α–helix of BSA from 65.4% to 51.2%. The melting temperature studies showed that these five sweeteners except REB-A act as stabilizers to increase the thermal stability of BSA during the thermal denaturation process. In addition, AK, TGS, and SC obviously increased the esterase-like activity of BSA, and such loss of activity of BSA induced by APM and REB-A.

Conformational and functional changes of bovine serum albumin induced by TiO2 nanoparticles binding

In the present study, we have investigated the thermal stability of bovine serum albumin (BSA) in the presence of TiO2 nanoparticles (TiO2 NPs). The effects of TiO2 NPs on the transportation function of BSA were also studied by fluorescence spectral method. The circular dichroism (CD) study revealed that addition of TiO2 NPs moderately decreased the α-Helix content of the protein with the abruptly changing its tertiary structure by hydrogen bonding, van der Waals or electrostatic forces. It has been found that TiO2 NPs acted as a structure destabilizer for BSA during the thermal denaturation process. The fluorescence quenching and molecular modeling data indicated the existence of strong interaction between BSA and the TiO2 NPs surface that changed the binding affinities of some ligands for BSA, implying that TiO2 NPs in blood will affect the transporting ability of serum albumin.

Comparison of thermal properties of fish collagen and bovine collagen in the temperature range 298–670 K

The increased interest in fish collagen is a consequence of the risk of exposure to Creutzfeld-Jacob disease (CJD) and the bovine spongiform encephalopathy (BSE), whose occurrence is associated with prions carried by bovine collagen.Collagen is the main biopolymer in living organisms and the main component of the skin and bones. Until the discovery of the BSE, bovine collagen had been widely used. The BSE epidemic increased the interest in new sources of collagen such as fish skin collagen (FSC) and its properties.Although the thermal properties of collagen originating from mammals have been well described, less attention has been paid to the thermal properties of FSC. Denaturation temperature is a particularly important parameter, depending on the collagen origin and hydration level. In the reported experiment, the free water and bound water release processes along with thermal denaturation process were studied by means of the differential scanning calorimetry (DSC). Measurements were carried out using a DSC 7 instrument (Elmer-Perkin), in the temperature range 298–670K. The study material was FSC derived by acidic hydration method. The bovine Achilles tendon (BAT) collagen type I was used as the control material. The thermograms recorded revealed both, exothermic and endothermic peaks. For both materials, the peaks in the temperature range of 330–360K were assigned to the release of free water and bound water. The denaturation temperatures of FSC and BAT collagen were determined as 420K and 493K, respectively. Thermal decomposition process was observed at about 500K for FSC and at about 510K for BAT collagen. These results show that FSC is less resistant to high temperature than BAT collagen.

Temperature-Induced Misfolding in Prion Protein: Evidence of Multiple Partially Disordered States Stabilized by Non-Native Hydrogen Bonds.

The structural basis of pathways of misfolding of a cellular prion (PrPC) into the toxic scrapie form (PrPSC) and identification of possible intermediates (e.g., PrP*) still eludes us. In this work, we have used a cumulative ∼65 μs of replica exchange molecular dynamics simulation data to construct the conformational free energy landscapes and capture the structural and thermodynamic characteristics associated with various stages of the thermal denaturation process in human prion protein. The temperature-dependent free energy surfaces consist of multiple metastable states stabilized by non-native contacts and hydrogen bonds, thus rendering the protein prone to misfolding. We have been able to identify metastable conformational states with high β-content (∼30-40%) and low α-content (∼10-20%) that might be precursors of PrPSC oligomer formation. These conformations also involve participation of the unstructured N-terminal domain, and its role in misfolding has been investigated. All the misfolded or partially unfolded states are quite compact in nature despite having large deviations from the native structure. Although the number of native contacts decreases dramatically at higher temperatures, the radius of gyration and number of intraprotein hydrogen bonds and contacts remain relatively unchanged, leading to stabilization of the misfolded conformations by non-native interactions. Our results are in good agreement with the established view that the C-terminal regions of the second and third helices (H2 and H3, respectively) of mammal prions might be the Achilles heels of their stability, while separation of B1-H1-B2 and H2-H3 domains seems to play a key role, as well.

ДЕТЕКТИРОВАНИЕ КОНФОРМАЦИОННЫХ ИЗМЕНЕНИЙ АЛЬБУМИНА ПРИ ЕГО ГЛИКИРОВАНИИ И ТЕРМИЧЕСКОЙ ДЕНАТУРАЦИИ МЕТОДАМИ ФЛУОРЕСЦЕНТНОЙ СПЕКТРОСКОПИИ И МАЛОУГЛОВОГО РЕНТГЕНОВСКОГО РАССЕЯНИЯ

ДЕТЕКТИРОВАНИЕ КОНФОРМАЦИОННЫХ ИЗМЕНЕНИЙ АЛЬБУМИНА ПРИ ЕГО ГЛИКИРОВАНИИ И ТЕРМИЧЕСКОЙ ДЕНАТУРАЦИИ МЕТОДАМИ ФЛУОРЕСЦЕНТНОЙ СПЕКТРОСКОПИИ И МАЛОУГЛОВОГО РЕНТГЕНОВСКОГО РАССЕЯНИЯ

Predicting the distribution of whey protein fouling in a plate heat exchanger using the kinetic parameters of the thermal denaturation reaction of β-lactoglobulin and the bulk temperature profiles.

Fouling of plate heat exchangers (PHE) is a severe problem in the dairy industry, notably because the relationship between the build-up of protein fouling deposits and the chemical reactions taking place in the fouling solution has not yet been fully elucidated. Experiments were conducted at pilot scale in a corrugated PHE, and fouling deposits were generated using a model β-lactoglobulin (β-LG) fouling solution for which the β-LG thermal denaturation reaction constants had been previously determined experimentally. Then 18 different bulk temperature profiles within the PHE were imposed. Analysis of the fouling runs shows that the dry deposit mass per channel versus the ratio R=kunf/kagg (with kunf and kagg representing, respectively, the unfolding and aggregation rate constants computed from both the identification of the β-LG thermal denaturation process and knowledge of the imposed bulk temperature profile into the PHE channel) is able to gather reasonably well the experimental fouling mass data into a unique master curve. This type of representation of the results clearly shows that the heat-induced reactions (unfolding and aggregation) of the various β-LG molecular species in the bulk fluid are essential to capture the trend of the fouling mass distribution inside a PHE. This investigation also illustrates unambiguously that the release of the unfolded β-LG (also called β-LG molten globule) within the bulk fluid (and the absence of its consumption in the form of aggregates) is a key phenomenon that controls the extent of protein fouling as well as its location inside the PHE.

In situ Detection of Amide A Bands of Proteins in Water by Raman Ratio Spectrum

The amide A band of protein is sensitive to the hydrogen bands of amide groups of proteins. However, it is hard to distinguish the amide A band of aqueous protein in situ directly, since it overlaps with O−H stretching vibration of water. In this work, we presented a new analytical method of Raman ratio spectrum, which can extract the amide A band of proteins in water. To obtain the Raman ratio spectrum, the Raman spectrum of aqueous protein was divided by that of pure water. A mathematical simulation was employed to examine whether Raman ratio spectrum is effective. Two kinds of protein, lysozyme and α-chymotrypsin were employed. The amide A bands of them in water were extracted from Raman ratio spectra. Additionally, the process of thermal denaturation of lysozyme was detected from Raman ratio spectrum. These results demonstrated the Raman ratio spectra could be employed to study the amide A modes of proteins in water.

Application of ATR-FTIR spectroscopy to the study of thermally induced changes in secondary structure of protein molecules in solid state.

FTIR spectroscopy in combination with ATR sampling technique is the most accessible analytical technique to study secondary structure of proteins both in solid and aqueous solution. Although several studies have demonstrated the applications of ATR-FTIR to study conformational changes of solid dried proteins due to dehydration, there are no reports that demonstrate the application of ATR-FTIR in the study of thermally induced changes of secondary structure of biomolecules directly on the solid state. In this study, four biomolecules of pharmaceutical interest, lysozyme, myoglobine, chymotripsin and human growth hormone (hGH), were studied on the solid state before and after different thermal treatments in order to relate changes of secondary structure to partial or total thermal denaturation processes. The results obtained provide experimental evidence that protein thermal denaturation in the solid state can be detected by displacement of carbonyl bands which correspond to conformational transformations between α-helix to β-sheet or intermolecular β-sheet; the molecules studied undergo this transformation when exposed to a temperature close to their denaturation temperature which may become irreversible depending on the extent of the heating treatment. These findings demonstrate that ATR-FTIR is an effective and time efficient technique that allows the monitoring of the protein thermal denaturation process of solid samples without further reconstitution or prior sample preparation.

Fourier transform infrared spectroscopy provides an evidence of papain denaturation and aggregation during cold storage

Papain is a cysteine protease with wide substrate specificity and many applications. Despite its widespread applications, cold stability of papain has never been studied. Here, we used differential spectroscopy to monitor thermal denaturation process. Papain was the most stabile from 45°C to 60°C with ΔG°321 of 13.9±0.3kJ/mol and Tm value of 84±1°C. After cold storage, papain lost parts of its native secondary structures elements which gave an increase of 40% of intermolecular β-sheet content (band maximum detected at frequency of 1621cm−1 in Fourier transform infrared (FT-IR) spectrum) indicating the presence of secondary structures necessary for aggregation. The presence of protein aggregates after cold storage was also proven by analytical size exclusion chromatography. After six freeze–thaw cycles around 75% of starting enzyme activity of papain was lost due to cold denaturation and aggregation of unfolded protein. Autoproteolysis of papain did not cause significant loss of the protein activity. Upon the cold storage, papain underwent structural rearrangements and aggregation that correspond to other cold denatured proteins, rather than autoproteolysis which could have the commercial importance for the growing polypeptide based industry.

Molecular insights into the role of aqueous trehalose solution on temperature-induced protein denaturation.

To investigate the underlying mechanism by which trehalose acts as a bioprotectant against thermal denaturation of protein in aqueous solution, we carry out classical molecular dynamics simulations at two different temperatures. Though it is widely accepted that trehalose acts as an antidote against such protein structural destabilization and numerous hypotheses have been proposed in regard to its mechanism of stabilization, there is still no definitive generally accepted answer to this question and it remains a subject of active research. In view of this, in this article we report the thermal denaturation process of a 15-residue S-peptide analogue at 360 K temperature and the counteracting ability of trehalose of varying concentrations at that temperature. In order to verify the conformational stability of the peptide at ambient temperature condition, we also carry out a separate simulation of peptide-water binary system at 300 K temperature. The goal is to provide a molecular level understanding of how trehalose protects protein at elevated temperature. The Cα-rmsd calculation shows that in pure water, the peptide is stable at 300 K temperature and its unfolding is observed at 360 K. However, in peptide-water-trehalose ternary system, the value of Cα-rmsd decreases as trehalose concentration is increased. Remarkably, at the highest trehalose concentration considered in this study, the value of Cα-rmsd at 360 K is similar to that of water-peptide binary system at 300 K temperature. Further, the calculations of radius of gyration of Cα-atoms and helical percentage of the peptide residues support the above observations. The total number of hydrogen bonds formed by the peptide with solution species (trehalose and water) remains constant, though the peptide water hydrogen bond decreases and peptide trehalose hydrogen bond increases with increasing trehalose concentration. This finding suggests replacement of water molecules by trehalose molecules and supports water replacement hypothesis. The calculations of preferential interaction parameter show that at the peptide surface, trehalose molecules are slightly more preferred over water and for the most concentrated solutions, a prominent exclusion of water and enrichment of trehalose molecules is observed. Also observed are (i) trehalose-induced second shell collapse of water structure, (ii) the growth of trehalose cluster as concentration is increased, and (iii) trehalose-induced slowing down of the translational motion of both water and trehalose, the effect being more pronounced for the latter. Implications of these results for counteracting mechanism of trehalose are discussed.

Relationship between β-relaxation and structural stability of lysozyme: microscopic insight on thermostabilization mechanism by trehalose from Raman spectroscopy experiments.

Raman investigations were carried out in the low-frequency and amide I regions on lysozyme aqueous solutions in absence and presence of trehalose. Raman spectroscopy gives the unique opportunity to analyze the protein and solvent dynamics in the low-frequency range while monitoring the unfolding process by capturing the spectrum of the amide I band. From the analysis of the quasielastic intensity, a dynamic change is firstly observed in a highly hydrated protein, around 70 °C, and interpreted in relation with the denaturation mechanism of the protein. The use of heavy water and partly deuterated trehalose gives clear information on protein-trehalose interactions in the native state of lysozyme (at room temperature) and during the thermal denaturation process of lysozyme. At room temperature, it was found that trehalose is preferentially excluded from the protein surface, and has a main effect on the tetrahedral local order of water molecules corresponding to a stiffening of the H-bond network in the solvent. The consequence is a significant reduction of the amplitude of fast relaxational motions, inducing a less marked dynamic transition shifted toward the high temperatures. Upon heating, interaction between trehalose and lysozyme is detected during the solvent penetration within the protein, i.e., while the native globular state softens into a molten globule (MG) state. Addition of trehalose reduces the protein flexibility in the MG state, improving the structural stability of the protein, and inhibiting the protein aggregation.

DESIGN OF <i>CANDIDA ANTARCTICA</i> LIPASE B THERMOSTABILITY IMPROVEMENT BY INTRODUCING EXTRA DISULFIDE BOND INTO THE ENZYME

Candida Antarctica Lipase B (CALB) is extensively studied in enzymatic production of biodiesel, pharmaceutical products, detergents and other chemi cals. One drawback of using CALB is its relatively low optimum temperature at 313 K (40°C). The objective of this research is to design CALB mutant with improved thermostability by introducing extra disul fide bond. Molecular dynamic simulation was conducted to get better insight into the process of thermal denaturation or unfolding in CALB. Thermal denaturation of CALB was accelerated by conducting simulation at high temperature. Molecular dynamic simulation of CALB was performed with GROMACS software package at 300-700 K. Prediction of possible mutation was done using “Disulfide by Desi gn TM ” software. Selection of mutated residues was based on flexibility analysis of CALB. From those a nalyses, three mutants were designed, which are Mutant-1 (73LeuCys/151AlaCys), Mutant-2 (155TrpCys/294GluCys) and Mutant-3 (43ThrCys/67SerCys). Parameters that were used to compare the thermostability of mutant with wild type enzyme were Root Mean Square Deviations (RMSD), Solvent Accessible Surface Area (SASA), Radius of gyration (Rg) and secondary structure. Molecular dynamic simulation conducted on those three mutants showed that Mutant-1 has better thermostability compared to wild type CA LB. We proposed the order of mutant thermostability improvement as follows: Mutant-1, Mutant-2 and Mutant-3, with Mutant-1 having better potential thermostability improvement and Mutant-3, the least stable.

Robust analysis of synthetic label-free DNA junctions in solution by X-ray scattering and molecular simulation

Structural analysis of branched DNA molecules (BDM) is important as model systems for DNA junctions and also as building units for DNA assembly. Although there have been efforts to study the structures of BDM, label-free solution structures have not been well determined yet. Here, we used a combination of synchrotron-based experimental tools and computational simulation to study the global structures of label-free BDM in solution. Overall structures of 3-arm and 4-arm BDM were revealed as an asymmetric T(or Y)-shape and a distorted X-shape, respectively. The internal structures of the DNA double helix were shown to have a canonical B-form for both the BDM. We also reconstructed the thermal denaturation process of BDM by determining the transient global structures over a wide range of temperatures. The proposed high-resolution structures of BDM are expected to provide fundamental information for studies of the biological function of junction DNAs and DNA assembly.

Investigation of thermal denaturation of solid bovine serum albumin by terahertz dielectric spectroscopy

We investigate the thermal denaturation of solid bovine serum albumin (BSA) using terahertz time domain spectroscopy (THz-TDS). When the protein is heated up from 25°C to 107°C and cooled down to 25°C again, an irreversible decrease in its THz absorption coefficient and refractive index is observed. The corresponding frequency-dependent permittivity during heating is fitted by the Debye model with single relaxation time. The relaxation times during temperature rising agree very well with Arrhenius equation with the activation energy of 6.52kJ/(K·mol), which can be an indicator for the stability of BSA during thermal denaturation process.

Interactions of the Human Telomere Sequence with the Nanocavity of the α-Hemolysin Ion Channel Reveal Structure-Dependent Electrical Signatures for Hybrid Folds

Human telomeric DNA consists of tandem repeats of the sequence 5'-TTAGGG-3', including a 3' terminal single-stranded overhang of 100-200 nucleotides that can fold into quadruplex structures in the presence of suitable metal ions. In the presence of an applied voltage, the α-hemolysin (α-HL) protein ion channel can produce unique current patterns that are found to be characteristic for various interactions between G-quadruplexes and the protein nanocavity. In this study, the human telomere in a complete sequence context, 5'-TAGGG(TTAGGG)3TT-3', was evaluated with respect to its multiple folding topologies. Notably, the coexistence of two interchangeable conformations of the K(+)-induced folds, hybrid-1 and hybrid-2, were readily resolved at a single-molecule level along with triplex folding intermediates, whose characterization has been challenging in experiments that measure the bulk solution. These results enabled us to profile the thermal denaturation process of these structures to elucidate the relative distributions of hybrid-1, hybrid-2, and folding intermediates such as triplexes. For example, at 37 °C, pH 7.9, in 50 mM aqueous KCl, the ratio of hybrid-1:hybrid-2:triplex is approximately 11:5:1 in dilute solution. The results obtained lay the foundation for utilizing the α-HL ion channel as a simple tool for monitoring how small molecules and physical context shift the equilibrium between the many G-quadruplex folds of the human telomere sequence.

A new aspect to chaperone-like activity of bovine β-casein by protein–protein interactions study

In the present work, chaperone-like activity of bovine β-casein (β-CN) against thermal denaturation and aggregation processes of bovine carbonic anhydrase (BCA) was investigated. We used different instrument and new developed methods for surveillance the structural alteration of BCA and its functional properties upon interaction with bovine β-CN. The thermodynamic data obtained by DSC showed that interaction of β-CN can enhance the thermal stability of enzyme but due to decrease of activation energy of aggregation, the kinetic of process as driven force accelerated the thermal aggregation. In the presence of β-CN due to enhancement of hydrophobicity and favoring the formation of first intermediate of CA, aggregation conveniently occurred with a higher rate at a low temperature.

Hysteresis in pressure-driven DNA denaturation.

In the past, a great deal of attention has been drawn to thermal driven denaturation processes. In recent years, however, the discovery of stress-induced denaturation, observed at the one-molecule level, has revealed new insights into the complex phenomena involved in the thermo-mechanics of DNA function. Understanding the effect of local pressure variations in DNA stability is thus an appealing topic. Such processes as cellular stress, dehydration, and changes in the ionic strength of the medium could explain local pressure changes that will affect the molecular mechanics of DNA and hence its stability. In this work, a theory that accounts for hysteresis in pressure-driven DNA denaturation is proposed. We here combine an irreversible thermodynamic approach with an equation of state based on the Poisson-Boltzmann cell model. The latter one provides a good description of the osmotic pressure over a wide range of DNA concentrations. The resulting theoretical framework predicts, in general, the process of denaturation and, in particular, hysteresis curves for a DNA sequence in terms of system parameters such as salt concentration, density of DNA molecules and temperature in addition to structural and configurational states of DNA. Furthermore, this formalism can be naturally extended to more complex situations, for example, in cases where the host medium is made up of asymmetric salts or in the description of the (helical-like) charge distribution along the DNA molecule. Moreover, since this study incorporates the effect of pressure through a thermodynamic analysis, much of what is known from temperature-driven experiments will shed light on the pressure-induced melting issue.

Terminal Amino Acids Disturb Xylanase Thermostability and Activity

Protein structure is composed of regular secondary structural elements (α-helix and β-strand) and non-regular region. Unlike the helix and strand, the non-regular region consists of an amino acid defined as a disordered residue (DR). When compared with the effect of the helix and strand, the effect of the DR on enzyme structure and function is elusive. An Aspergillus niger GH10 xylanase (Xyn) was selected as a model molecule of (β/α)(8) because the general structure consists of ~10% enzymes. The Xyn has five N-terminal DRs and one C-terminal DR, respectively, which were deleted to construct three mutants, XynΔN, XynΔC, and XynΔNC. Each mutant was ~2-, 3-, or 4-fold more thermostable and 7-, 4-, or 4-fold more active than the Xyn. The N-terminal deletion decreased the xylanase temperature optimum for activity (T(opt)) 6 °C, but the C-terminal deletion increased its T(opt) 6 °C. The N- and C-terminal deletions had opposing effects on the enzyme T(opt) but had additive effects on its thermostability. The five N-terminal DR deletions had more effect on the enzyme kinetics but less effect on its thermo property than the one C-terminal DR deletion. CD data showed that the terminal DR deletions increased regular secondary structural contents, and hence, led to slow decreased Gibbs free energy changes (ΔG(0)) in the thermal denaturation process, which ultimately enhanced enzyme thermostabilities.

Thermal denaturation of recombinant human lysozyme from rice: effect of pH and comparison with human milk lysozyme

Thermal denaturation of recombinant human lysozyme from rice has been studied by differential scanning calorimetry at acidic (4.2), neutral (7.2) and basic (9.2) pH levels at various heating rates, and it has been compared with thermal denaturation of human lysozyme isolated from milk at the same pH levels at a heating rate of 10 °C/min. Data obtained from heat-induced unfolding and subsequent refolding after heating indicate that thermal denaturation of both lysozymes undergoes a two-state process. The maximum temperature of the endothermic peaks and the enthalpy change of denaturation indicate that recombinant and milk lysozymes possess similar thermostability, which is higher at acidic than at neutral pH. On the other hand, both proteins are more thermolabile at basic pH. Lysozyme from human milk shows a higher tendency to aggregate than recombinant human lysozyme from rice during the thermal denaturation process.