Study Protein Denaturation Research Articles

Application of computational fluid dynamics simulation in predicting food protein denaturation: numerical studies on selected food products - a review*

Abstract Protein denaturation is a common process in the food industry, which can impact food quality and safety. Computational Fluid Dynamics (CFD) is a powerful tool that can be used to predict protein denaturation in food products. In this review article, we present an overview of the application of CFD simulation in predicting protein denaturation in food products. We discuss the factors that influence protein denaturation, the importance of predicting protein denaturation, and the various numerical methods used in protein denaturation studies. The main focus of the article is the use of CFD simulation in predicting protein denaturation in selected food products, such as milk, meat, and eggs. We provide examples of numerical studies that have been conducted on these products, and we discuss the results and implications of these studies. The use of CFD simulation can help to optimize food processing conditions, improve food quality and safety, and reduce waste and costs in the food industry. Overall, this review article highlights the importance of using advanced computational tools such as CFD simulation in food science, research and development. Highlights The use of CFD simulation can predict protein denaturation in food products. Numerical studies were conducted on selected food products to analyze protein denaturation. CFD simulation provides a powerful tool for optimizing food processing technologies. The results from the numerical studies can be used to improve the quality and safety of food products. The application of CFD simulation can lead to more efficient and sustainable food production practices.

In Vitro Anti Inflammatory, Anti-Oxidant and Anti-Cancer Cell Line Studies on Mollugo Cerviana (L.) Ser

Our traditional treatises on medicine have shown references to number of rare medicinal herbs prescribed to treat various ailments in human. An herbal plant preferred to treat fever, inflammation, rheumatic pain and wounds prevalently in the villages of the delta districts of Tamilnadu by name Mollugo cerviana was chosen for this research. The Ethanol extract of Mollugo cerviana was analysed for its in vitro anti-inflammatory, antioxidant and anticancer activity through standard procedures. The anti-inflammatory activity of the ethanol ethanolic extracts of varying concentrations were evaluated under HRBC (Human Red Blood Cell) Membrane Stabilization and Protein denaturation studies. Diclofenac sodium a nonsterioidal anti-inflammatory drug was the reference standard. The results revealed a concentration dependent increase in the percentage of membrane stabilization activity with increase in concentration of the test extract and a concentration dependent inhibition of protein (albumin) denaturation activity. Antioxidant studies revealed a significant free radical scavenging activity by the extract upon evaluation by DPPH free radical scavenging assay, nitric oxide assay and hydrogen peroxide radical scavenging activity assay. The studies on liver cancer cell lines using MTT assay revealed the anticancer activity exhibited by the ethanol extract of Mollugo cerviana. The studies as a whole reveals the anti-inflammatory, antioxidant and anticancer activity possessed by the herbal extract of Mollugo cerviana which invites attention to proceed with further research towards development of novel herbal drugs from Mollugo cerviana to treat inflammation and cancer.

A molecular dynamics study of protein denaturation induced by sulfonate-based surfactants.

Microsecond timescale explicit-solvent atomistic simulations were carried out to investigate how anionic surfactants modulate protein structure and dynamics. We found that lysozyme undergoes near-complete denaturation at the high concentration (> 0.1M) of sodium pentadecyl sulfonate (SPDS), while only partial denaturation occurs at the concentration slightly below 0.1M. In large part, protein denaturation is structurally manifested by disappearance of helical segments and loss of tertiary interactions. The computational prediction of the extent of burial of cysteine residues was experimentally validated by measuring the accessibility of the respective sulfhydryl groups. Overall, our work indicates an interesting synergy between electrostatic and hydrophobic contributions to lysozyme's denaturation process by anionic surfactants. In fact, first disulfide bridges and hydrogen bonds from protein surface to SPDS head groups loosen the protein globule followed by fuller denaturation via insertion of the surfactant's hydrophobic tails into the protein core.

Studies of intrinsic volumes of ethylene glycol and polyethylene glycol–300 molecules in aqueous solutions at 298.15 K: Application of Tammann–Tait–Gibson (TTG) equation of state

Systemic density (ρ) measurements have been carried–out for aqueous solutions of polyethylene glycols (PEG's) with the molar mass 200, 300, 400 and 600 D at (288.15, 293.15, 298.15, 303.15 and 308.15) K and at ambient pressure of 0.1 MPa in dilute solute concentration region (0.0119–0.4941 mol⋅kg−1). The ρ data are used to obtain the apparent molar volume (Vϕ) and partial molar volume (V¯2) of studied PEG's in aqueous solutions at finite concentration as well as at infinitely dilute solutions (Vϕ0) at studied temperatures. The coefficient of thermal expansion (α) for solutions, apparent molar expansivity (Eϕ) and limiting apparent molar expansivity (Eϕ0) for solutes are calculated at 293.15, 298.15 and 303.15 K. The densities of studied PEG's in carbon tetrachloride (CCl4) medium are also measured at 298.15 K. The transfer volume (ΔtrVϕ0) for PEG from pure liquid state to CCl4 and to water as solvent are estimated. The literature data of sound velocity (u), coefficient of thermal expansion (α) and specific heat capacity (CP) for solutions have been utilized to obtain thermodynamic properties like adiabatic compressibility (βad) and isothermal compressibility (βT) in case of aqueous solutions of ethylene glycol (EG) and PEG–300 at 298.15 K. The data are further utilized to obtain apparent molar adiabatic compressibility (KϕS) and apparent molar isothermal compressibility (KϕT) for EG and PEG–300 solute molecules at finite and at infinitely dilute solute concentration (KϕS0 and KϕT0, respectively). Further, the data of KϕT, α and CP data have been utilized to estimate internal pressure Pi (=∂U∂VT) of solutions containing EG and PEG–300 molecules as a function of concentration. The intrinsic volumes (Vint.) of these molecules have been calculated by applying Tammann–Tait–Gibson (TTG) equation of state. It is being observed that intrinsic volume differs appreciably from the value based upon EG and PEG–300 in a liquid state as a standard state. The excess volume change (V¯20E=V¯20-V¯int.0) values are found to be −16.1 and −63.8⋅10–6 m3⋅mol−1 for EG and PEG–300, respectively. Similar calculations made for PEG–300 in CCl4 medium also yield excess volume change of −60⋅10–6 m3⋅mol−1 magnitude. These results are explained on the basis of three dimensional hydrogen bonded structure of EG and the linkage ▪ interaction with solvent water having a presence of helical segments. The inferences drawn will be of help to understand the weak denaturing interactions of glycols in protein denaturation studies.

Synthesis and Anti-inflammatory Activity of 7-Non-steroidal Anti-inflammatory Drugs Substituted Flavone

Aim: Inflammation shows an important character in illnesses such for example cardiovascular diseases, asthma, malignant and diabetes. Food can guidance different phases of inflammation and can have an important influence on numerous inflammatory illnesses. Increasing scientific suggestion has displayed that polyphenolic compounds, such as flavonoids, which are originate in vegetables, fruits, cocoa or legumes, can have antiinflammatory activity. Current researches prevent enzymes or transcription factors significant for regulatory intermediaries involved in inflammation. Flavonoids are also famous as effective antioxidants with the potential to weaken tissue injury or fibrosis. Subsequently, frequent studies in vivo and in vitro models shows that flavonoids have the prospective to prevent the beginning and development of inflammatory activity. Materials and Methods: In the present research work, a series of flavones merge with Non-steroidal Anti-Inflammatory Drugs were synthesized from resorcinol through Baker- Venkataraman Method, characterized and calculated there in vitro anti-inflammatory activity by protein denaturation technique. Results: The outcome is protein denaturation studies shown that the compound FS found to be most potent anti-inflammatory activity. The percentage inhibitions were 97.66%. Key words: Albumin Denaturation, Baker Venkataraman transformation, Cyclooxygenase, Flavone, NSAIDS.

Lipid composition and macromolecular crowding effects on CYP2J2-mediated drug metabolism in nanodiscs.

Lipid composition and macromolecular crowding are key external effectors of protein activity and stability whose role varies between different proteins. Therefore, it is imperative to study their effects on individual protein function. CYP2J2 is a membrane-bound cytochrome P450 in the heart involved in the metabolism of fatty acids and xenobiotics. In order to facilitate this metabolism, cytochrome P450 reductase (CPR), transfers electrons to CYP2J2 from NADPH. Herein, we use nanodiscs to show that lipid composition of the membrane bilayer affects substrate metabolism of the CYP2J2-CPR nanodisc (ND) system. Differential effects on both NADPH oxidation and substrate metabolism by CYP2J2-CPR are dependent on the lipid composition. For instance, sphingomyelin containing nanodiscs produced more secondary substrate metabolites than discs of other lipid compositions, implying a possible conformational change leading to processive metabolism. Furthermore, we demonstrate that macromolecular crowding plays a role in the lipid-solubilized CYP2J2-CPR system by increasing the Km and decreasing the Vmax , and effect that is size-dependent. Crowding also affects the CYP2J2-CPR-ND system by decreasing both the Km and Vmax for Dextran-based macromolecular crowding agents, implying an increase in substrate affinity but a lack of metabolism. Finally, protein denaturation studies show that crowding agents destabilize CYP2J2, while the multidomain protein CPR is stabilized. Overall, these studies are the first report on the role of the surrounding lipid environment and macromolecular crowding in modulating enzymatic function of CYP2J2-CPR membrane protein system.

Fast scanning calorimetry of lysozyme unfolding at scanning rates from 5 K/min to 500,000 K/min

BackgroundProtein denaturation is often studied using differential scanning calorimetry (DSC). However, conventional instruments are limited in the temperature scanning rate available. Fast scanning calorimetry (FSC) provides an ability to study processes at much higher rates while using extremely small sample masses [ng]. This makes it a very interesting technique for protein investigation. MethodsA combination of conventional DSC and fast scanning calorimeters was used to study denaturation of lysozyme dissolved in glycerol. Glycerol was chosen as a solvent to prevent evaporation from the micro-sized samples of the fast scanning calorimeter. ResultsThe lysozyme denaturation temperatures in the range of scanning rates from 5 K/min to ca. 500,000 K/min follow the Arrhenius law. The experimental results for FSC and conventional DSC fall into two distinct clusters in a Kissinger plot, which are well approximated by two parallel straight lines. ConclusionsThe transition temperatures for the unfolding process measured on fast scanning calorimetry sensor are significantly lower than what could be expected from the results of conventional DSC using extrapolation to high scanning rates. Evidence for the influence of the relative surface area on the unfolding temperature was found. General significanceFor the first time, fast scanning calorimetry was employed to study protein denaturation with a range of temperature scanning rates of 5 orders of magnitude. Decreased thermal stability of the micro-sized samples on the fast scanning calorimeter raise caution over using bulk solution thermal stability data of proteins for applications where micro-sized dispersed protein solutions are used, e.g., spray drying.

Intrinsic Fluorescence as a Spectral Probe for Protein Denaturation Studies in the Presence of Honey

Honey was found to quench the intrinsic fluorescence of bovine serum albumin (BSA) in a concentration dependent manner, showing complete quenching in the presence of 5% (w/v) honey. Increasing the protein concentration up to 5.0 μM did not lead to the recovery of the protein fluorescence. Urea denaturation of BSA, which otherwise shows a two-step, three-state transition, using intrinsic fluorescence of the protein as the probe failed to produce any result in the presence of 5% (w/v) honey. Thus, intrinsic fluorescence cannot be used as a spectral probe for protein denaturation studies in the presence of honey.

Anti-tumour-promoting and thermal-induced protein denaturation inhibitory activities of β-sitosterol and lupeol isolated from Diospyros lotus L.

In this study, the anti-tumour-promoting and thermal-induced protein denaturation inhibitory activities of β-sitosterol (1) and lupeol (2), isolated from Diospyros lotus L., were explored. Compound 1 showed a marked concentration-dependent inhibition against 12-O-tetradecanoylphorbol-13-acetate (20 ng/32 pmol)-induced Epstein–Barr virus early antigen activation in Raji cells with IC50 of 270 μg/ml, without significant toxicity (70% viability). Compound 2 showed significant anti-tumour-promoting effect with IC50 of 412 μg/ml, without significant toxicity (60% viability). In heat-induced protein denaturation assay, compound 1 exhibited a concentration-dependent attenuation with a maximum effect of 73.5% at 500 μg/ml with EC50 of 117 μg/ml, while compound 2 exhibited a maximum effect of 59.2% at 500 μg/ml with EC50 of 355 μg/ml. Moreover, in silico docking studies against the phosphoinositide 3-kinase enzyme also show the inhibitory potency of these compounds. In short, both the compounds exhibited a marked anti-tumour-promoting and potent inhibitory effect on thermal-induced protein denaturation.

Phasor approaches simplify the analysis of tryptophan fluorescence data in protein denaturation studies

The intrinsic fluorescence of tryptophan is frequently used to investigate the structure of proteins. The analysis of tryptophan fluorescence data is challenging: fluorescence (anisotropy) decays typically have multiple lifetime (correlation time) components and fluorescence spectra are broad and exhibit only minor shifts. In this work, we show that phasor approaches can substantially simplify tryptophan fluorescence analysis. To demonstrate this, we re-analyse previously recorded datasets of the denaturant (guanidinium hydrochloride, GuHCl) induced unfolding of a single-tryptophan-containing variant of apoflavodoxin from Azotobacter vinelandii. For three methods—(1) time-resolved fluorescence, (2) time-resolved fluorescence anisotropy and (3) steady-state fluorescence spectroscopy—we show that the phasor analysis can readily identify the presence of a folding intermediate. Moreover, the fractional contributions of protein states at various stages of unfolding and the values of the free energy difference of the unfolding process are obtained. The outcomes are compared to the global analysis results published previously.

Phasor approaches simplify the analysis of tryptophan fluorescence data in protein denaturation studies

Phasor approaches simplify the analysis of tryptophan fluorescence data in protein denaturation studies

Use of FCS to Study Protein Denaturation and Aggregation

Fluorescence Correlation Spectroscopy (FCS) is a powerful technique for studying the diffusive dynamics of fluorophore-tagged biomolecules and thereby drawing inferences about their conformational states and transitions. We have employed FCS to investigate the denaturation and aggregation pathways of bovine immunoglobulin (IgG), a multi-subunit all beta-sheet Y-shaped protein prone to aggregation. Each upper arm of the Y consists of a light and a heavy chain connected by cysteine-shy;cysteine disulphide bonds, and the two heavy chains are joined at the bend by more disulphides. While majority of proteins show continuous increase of hydrodynamic radius (Rg) when presented with increasing denaturant concentration, IgG exhibits strikingly different behavior under similar conditions. Its Rg initially decreases with increasing concentration of GdnHCl (from 0 to 3 M) and then goes through two pronounced maxima at 4 M and 7 M [GdnHCl]. We propose that the initial contraction is related to sheet-to-helix transition at the flexible bends of the Y-arms of the protein. The first maximum of Rg is attributed to electrostatic repulsions and their screening by Gdn+ and Cl- ions; the second is shown to arise from disulphide-mediated effects.Previous studies have proposed that IgG aggregation is initiated by intermolecular disulphide bond formation. We studied the aggregation kinetics of IgG by monitoring the time evolution of characteristic diffusion time (τD) under two different conditions: with the cysteine residues free to interact or blocked. The progress curves (τD vs.elapsed time) were sigmoidal in both cases, but the delay time was almost an order of magnitude longer, and the apparent polymerization rate half as fast, for the case where the cysteines were blocked than when they were free. This result indicates that intermolecular disulphide bond formation is a key factor driving the aggregation process of IgG.

Unified Description of Urea Denaturation: Backbone and Side Chains Contribute Equally in the Transfer Model

After studying protein denaturation by urea for many decades, conflicting views of the role of the side chains and the backbone have emerged; many results suggest that urea denatures by enhancing the solubility of both the side chains and the backbone, but the frequently applied transfer model (TM) so far ascribes denaturation exclusively to urea's action on the backbone. We use molecular dynamics simulations to rigorously test one of the TM's key assumptions, the proportionality of a molecule's transfer free energy (TFE) and its solvent-accessible surface. The performance of the TM as it is usually implemented turns out to be unsatisfactory, but the proportionality is satisfied very well after an inconsistency in the treatment of the backbone contribution is corrected. This inconsistency has so far gone unnoticed as it was obscured by a compensating error in the side-chain group TFEs used so far. The revised "universal backbone" TM presented in this work shows excellent accuracy in the prediction of experimental m values of a set of 36 proteins. It also settles the conflicting views regarding the role of the side chains because it predicts that both the side chains and the backbone on average contribute favorably to denaturation by urea.

Combining Small-angle Neutron and X-ray Scattering for Studying Protein Denaturation

Biological small-angle scattering (SAS) probes scattering-length density fluctuations of bio-macromolecules in solution that are of the order of nanometres (1–3). A new generation of modelling programs has allowed providing low-resolution envelopes of proteins with unknown high-resolution structure or rigid-body modelling of complexes that are composed of subunits of known high-resolution structure (4–6). Small-angle neutron scattering (SANS) can provide complementary structural information to small-angle X-ray scattering (SAXS) when the hydrogen densities vary between partners constituting a macromolecular complex (proteins, RNA, DNA, lipids …) and the aqueous solvent. This natural contrast can be modulated or enhanced by using deuteration of specific partners and/or by using heavy water (D2O) in the solvent. A thorough and comprehensive monograph on biological SANS and contrast variation has been written by Jacrot (7).

Denaturation Behaviors of Two-State and Non-Two-State Proteins Examined by an Interruption–Incubation Protocol

We established an interruption-incubation protocol to study the denaturation and aggregation behaviors of two-state and non-two-state proteins, represented by hen egg-white lysozyme (lysozyme) and bovine serum albumin (BSA), by differential scanning calorimetry (DSC) and Fourier-transform infrared (FTIR) spectroscopy. After incubation at selected interrupting temperatures (T(int)), the onset temperature (T(onset)) and denaturation temperature (T(d)) of the reheating scans were found to pose distinct contrasts between the two proteins. BSA shows increasing T(onset) and T(d) as T(int) increases, whereas lysozyme exhibits invariable T(onset) and T(d). After long-time incubation, the reheating scans of BSA still show residual peaks in the DSC curves. Moderate aggregation upon incubation restricts the refolding of the thermodynamically stable unfolding intermediates in the denaturation of BSA. On the contrary, prolonged incubation at selected T(int) causes complete denaturation of lysozyme. The results were also supported by FTIR experiments. It is concluded that the variable T(onset) and T(d) indicate the unfolding of the "trapped" intermediates in the second heating scans, and the invariable T(onset) and T(d) values indicate the nonexistence of such intermediates. The examination of two additional proteins, ribonuclease A and γ-globulin, shows a similar phenomenon. The results may represent the general features of two-state and non-two-state denaturations and the protocol would serve to study protein denaturation cooperativity.

Measurement of circular dichroism dynamics in a nanosecond temperature-jump experiment

The use of a fast temperature jump (T-jump) is a very powerful experiment aiming at studying protein denaturation dynamics. However, probing the secondary structure is a difficult challenge and rarely yields quantitative values. We present the technical implementation of far-UV circular dichroism in a nanosecond T-jump experiment and show that this experiment allows us to follow quantitatively the change in the helical fraction of a poly(glutamic acid) peptide during its thermal denaturation with 12 ns time resolution.

Equilibrium Studies of Protein Denaturation by Urea - Examining Backbone and Sidechain Contributions

Urea is a commonly used denaturant in protein folding studies, and it is of great interest to determine its interaction with various protein groups to elucidate the molecular basis of its effect on protein stability. Using the Trp-cage miniprotein as a model system, we report changes in preferential interaction coefficient of the protein upon urea denaturation and examine the contribution from the backbone and sidechain groups. The preferential interaction was obtained from reversible folding/unfolding Replica Exchange MD simulations of Trp-cage in presence of urea, over a broad range of urea concentration. The simulations were carried out using two different forcefields. AMBER94 and AMBER99sb, in conjunction with TIP3P water and KBFF urea models. In a given ensemble, folded or unfolded, we find the sidechains have a larger preferential interaction than that of the backbone. The increase in preferential interaction upon unfolding can be accounted entirely by sidechain contributions in AMBER94 simulations. In AMBER99sb, we find that both backbone and sidechain contribute to the increase in preferential interaction, with the sidechain contribution being slightly larger. The differences between two forcefields can be attributed to the unfolded ensemble sampled, with AMBER99sb favoring conformations with larger surface area and lower helical content. Both forcefields show that the preferential binding is dominated by binding of urea to sidechain atoms. This work has been supported by the National Science Foundation (Grant MCB-0543769).

Equilibrium Study of Protein Denaturation by Urea

Though urea is commonly used to denature proteins, the molecular mechanism of its denaturing ability is still a subject of considerable debate. Previous molecular dynamics simulation studies have sought to elucidate the mechanism of urea denaturation by focusing on the pathway of denaturation rather than examining the effect of urea on the folding/unfolding equilibrium, which is commonly measured in experiment. Here we report the reversible folding/unfolding equilibrium of Trp-cage miniprotein in the presence of urea, over a broad range of urea concentrations, using all-atom Replica exchange MD simulations. The simulations capture the experimentally observed linear dependence of unfolding free energy on urea concentration. We find that the denaturation is driven by favorable direct interaction of urea with the protein through both electrostatic and van der Waals forces and quantify their contribution. Though the magnitude of direct electrostatic interaction of urea is larger than van der Waals, the difference between unfolded and folded ensembles is dominated by the van der Waals interaction. We also find that hydrogen bonding of urea to the peptide backbone does not play a dominant role in denaturation. The unfolded ensemble sampled depends on urea concentration, with greater urea concentration favoring conformations with greater solvent exposure. The m-value is predicted to increase with temperature and more strongly so with pressure.

Zinc Porphyrin as a Donor for FRET in Zn(II)cytochrome c

We demonstrate that Zn(II) porphyrin in Zn(II)cytochrome c (Zn cyt c) is a fluorescence resonance energy transfer (FRET) donor to an Alexa660 dye acceptor. The energy transfer efficiency is dependent on the distance between the two fluorophores as shown through protein denaturation studies of five Zn cyt c variants labeled with Alexa660 in different positions. The relative quantum yield, excitation and emission energies, and labeling efficiencies of this donor-acceptor pair allow for a method of analysis based on sensitized emission of the acceptor. These studies show that Zn(II) porphyrin is an effective energy donor for measurement of molecular-scale distances by FRET.

Borrowing a little from research to enhance undergraduate teaching

Faculty members in typical teaching and research universities constantly feel pulled in three different directions –teaching, research and service. These constant demands and tug-of-war often leads us to unfortunately “cut corners” and not take our teaching as seriously and instead make us focus on our research activities. However, with a little bit of work and creativity, it is possible that we can “borrow a little from our various research activities to enhance our undergraduate teaching”. This poster will present three examples where the author has taken basic elements of three different biochemistry research projects and incorporated them into his undergraduate biochemistry laboratory classes. This not only gives students the opportunity to see latest topics in the faculty members’ research but to also see how chemistry (or biochemistry) actually addresses real-world issues and concerns. The three topics deal with 1) using GFP to study protein denaturation, 2) using Alkaline Phosphatase assays to study basic enzyme kinetics, and 3) use of Advanced Oxidation Processes to degrade organic pollutants. Experimental set-ups of each of these three undergraduate experiments will be presented in detail.