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Protein unfolding is a ubiquitous process responsible for the loss of protein functionality (denaturation), which, in turn, can be accompanied by the death of cells and organisms. The nature of enthalpy–entropy compensation (EEC) in the kinetics of protein unfolding is a subject of debate. In order to investigate the nature of EEC, the "completely loose" transition state (TS) model has been applied to calculate the Arrhenius parameters for the unfolding of polyglycine dimers as a model process. The calculated Arrhenius parameters increase with increasing dimer length and demonstrate enthalpy–entropy compensation. It is shown that EEC results from the linear correlations of enthalpy and entropy of activation with dimer length, which are derived directly from the properties of the transition state. It is shown that EEC in solvated (hydrated, etc.) proteins is a direct consequence of EEC in proteins themselves. The suggested model allows us also to reproduce and explain "exotic" very high values of the pre-exponential factor measured for the proteins unfolding, which are drastically higher than those known for unimolecular reactions of organic molecules. A similar approach can be applied to analyzing the nature of EEC phenomena observed in other areas of chemistry. [ABSTRACT FROM AUTHOR]

The limited water solubility and bioactivity of hydrophobic phytochemicals can be improved and preserved by encapsulation technologies. In this research, a low-cost and low-energy encapsulation method was explored based on the self-assembly characteristics of soy protein (SP). The disulfide bond of SP was broken by Na 2 S 2 O 5 , and then the pH of SP was adjusted to the critical pH 10.5 with NaOH, which was the critical point of protein unfolding. The treated protein was added to curcumin (Cur), which was encapsulated in self-assembled protein nanoparticles during the subsequent neutralization process to obtain the soy protein-curcumin complex (SP-Cur). This process was known as the disulfide bond breaking combined with critical pH-driven technology. The cleavage of SP disulfide bonds was studied by the methods of 4, 4′-dithiopyridine (DPS) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). When the SP:Cur mass ratio was 1:1, the encapsulation efficiency (EE%) of Cur in SP-Cur obtained by disulfide bond breaking combined with the critical pH-driven method increased to 85.36 ± 1.03%. It was notably greater than the EE% of Cur in SP-Cur prepared by the pH-driven method (61.82 ± 1.54%) and the EE% of Cur in SP-Cur prepared by disulfide bond breaking treatment (66.38 ± 1.2%). The encapsulation of Cur in SP particles exhibited significant resistance to light and temperature, leading to enhanced stability. Soy protein treated with disulfide bond cleavage combined with critical pH-driven method can serve as a potential functional carrier to further incorporate hydrophobic compounds into food systems. [Display omitted] • A low-cost and low-energy encapsulation method was explored. • Combined technology could achieve high encapsulation efficiency of curcumin. • •Combined technology was more effective than single technology. [ABSTRACT FROM AUTHOR]

• The study analyzed fenugreek α-amylase denaturation under different pH, temperature, and denaturant conditions. • Protein folding kinetics and stability were analyzed using fluorescence, circular dichroism, Raman spectroscopy, and enzyme assays. • The enzyme remained stable up to 70 °C but aggregated at higher temperatures. • The enzyme remained structurally and functionally stable between pH 4.0–8.0, with 88 % hydrolytic activity recovery. • Urea and GdHCl-induced denaturation showed irreversible, non-coincidental cooperative changes in α-amylase unfolding, contradicting prior two-state process findings. Fenugreek (Trigonella foenum-graecum) α-amylase is a protein with multiple domains of significant importance in various industrial applications. This enzyme, with a natural molecular weight of 47.5 kDa, is used in starch-based industries because of its strong characteristics and effective ability to break down molecules. This work aims to investigate the denaturation pathways and unfolded states of α-amylase from fenugreek under various environmental circumstances such as pH, temperature, and denaturants (chemical). The protein exhibits distinct unfolding paths in various contexts, serving as a paradigmatic illustration of how the environment determines the folding trajectory of a protein, whereas its amino acid sequence only determines its ultimate three-dimensional shape. The results of our investigation into α-amylase unfolding, conducted using a variety of analytical techniques such as fluorescence, circular dichroism, Raman spectroscopy, and enzyme assays, provide valuable insights into the protein's folding dynamics and resilience. The enzyme displayed exceptional stability up to 70 °C but tended to aggregate at higher temperatures. Partial unfolding occurred under acidic conditions, indicating that the enzyme retained some of its structural integrity. Within the pH range of 4.0–8.0, the enzyme remained both structurally and functionally stable, with an 88 % recovery of hydrolytic activity. Our investigation into the biophysical characteristics of intermediate stages during urea and GdHCl-induced denaturation revealed that α-amylase unfolding involves irreversible and non-coincidental cooperative transitions. This differs from previous findings of a two-state unfolding mechanism. The presence of a molten globule state was found in the study of the enzyme and confirmed by various spectroscopic analysis. The molten globule state being an intermediate stage in the unfolding of a protein deviates from the two-state theory. Through these sequential investigative steps, our study provides a comprehensive understanding of the stability and folding dynamics of α-amylase, offering valuable insights for its biotechnological applications. These findings provide a deeper understanding of the structural nuances of α-amylase and their correlation with its catalytic performance. Our work offers insights into the structural characteristics of α-amylase and how they are related to its catalytic activity. Understanding the stability and folding characteristics of α-amylase will improve biotechnological applications due to its significant role in starch-based industries. [ABSTRACT FROM AUTHOR]

To elucidate the roles of interfacial protein microstructure in regulating techno-functional attributes of complex microgel, this work explored the effects of transglutaminase cross-linking alone or in combination with ultrasonication on conformation and functional properties of whey protein isolate-chitooligosaccharide microgel dispersion (WPI–COS). It was found that transglutaminase-crosslinked WPI-COS microgels presented improved interfacial properties when a moderate unfolding process was induced by sonication, validating the existence of optimum flexibility of complex Pickering particles during unfolding/refolding of interfacial proteins. Particularly, TG induced excessive crosslinking of WPI-COS to form aggregates, causing a lower surface hydrophobicity. In contrast, ultrasound was inclined to increase the surface hydrophobicity for TG-catalyzed and uncatalyzed samples, suggesting differential unfolding degrees of WPI induced by transglutaminase and ultrasonication. Correspondingly, interfacial properties of ultrasonicated WPI-COS complexes, as revealed by percentage of adsorbed protein, interfacial tension and three-phase contact angle and emulsifying properties, were improved significantly. Confocal laser scanning microscopy and transmission electron microscopy consistently observed WPI-COS tightly adsorbed at the oil-water interface, with smaller emulsion droplets, more uniform distribution and thicker interfacial films at the oil-water interface in WPI-COS microgel particles with sonication-induced unfolding. This investigation demonstrated the feasibility of conformational regulation of interfacial proteins in tuning techno-functional features of microgel particles. [Display omitted] • Transglutaminase (TG) coupled to sonication was used to fabricate microgel particle. • TG catalyzed excessive crosslinking of WPI-COS to form aggregates. • Sonication-induced unfolding enhanced the flexibility of microgel particles. • Ultrasound-induced unfolding improved interfacial properties of WPI-COS microgels. • Moderate unfolding counteracted adverse effects of TG-induced WPI aggregation. [ABSTRACT FROM AUTHOR]

The functional structure of proteins results from marginally stable folded conformations. Reversible unfolding, irreversible denaturation, and deterioration can be caused by chemical and physical agents due to changes in the physicochemical conditions of pH, ionic strength, temperature, pressure, and electric field or due to the presence of a cosolvent that perturbs the delicate balance between stabilizing and destabilizing interactions and eventually induces chemical modifications. For most proteins, denaturation is a complex process involving transient intermediates in several reversible and eventually irreversible steps. Knowledge of protein stability and denaturation processes is mandatory for the development of enzymes as industrial catalysts, biopharmaceuticals, analytical and medical bioreagents, and safe industrial food. Electrophoresis techniques operating under extreme conditions are convenient tools for analyzing unfolding transitions, trapping transient intermediates, and gaining insight into the mechanisms of denaturation processes. Moreover, quantitative analysis of electrophoretic mobility transition curves allows the estimation of the conformational stability of proteins. These approaches include polyacrylamide gel electrophoresis and capillary zone electrophoresis under cold, heat, and hydrostatic pressure and in the presence of non-ionic denaturing agents or stabilizers such as polyols and heavy water. Lastly, after exposure to extremes of physical conditions, electrophoresis under standard conditions provides information on irreversible processes, slow conformational drifts, and slow renaturation processes. The impressive developments of enzyme technology with multiple applications in fine chemistry, biopharmaceutics, and nanomedicine prompted us to revisit the potentialities of these electrophoretic approaches. This feature review is illustrated with published and unpublished results obtained by the authors on cholinesterases and paraoxonase, two physiologically and toxicologically important enzymes. [ABSTRACT FROM AUTHOR]

Pharmacogenomics is a powerful tool to prevent adverse reactions caused by different response of individuals to drug administration. Single nucleotide polymorphisms (SNPs) represent up to 90% of genetic variations among individuals. Drug metabolizing enzymes are highly polymorphic therefore the kinetic parameters of their catalytic reactions can be significantly influenced. This work reports on the unfolding process of a phase I drug metabolizing enzyme, human flavin-containing monooxygenase 3 (hFMO3) and its single nucleotide polymorphic variants (SNPs) V257M, E158K and E308G. Differential scanning calorimetry (DSC) indicates that the thermal denaturation of the enzyme is irreversible. The melting temperature (T m) for the (Wild Type) WT and its polymorphic variants is found to be in a range from 46 °C to 50 °C. Also the activation energies of unfolding (E a) show no significant differences among all proteins investigated (290–328 KJ/mol), except for the E308G variant that showed a significantly higher E a of 412 KJ/mol. The presence of the bound NADP+ cofactor is found to stabilize all the variants by shifting the main T m by 4–5 °C for all the proteins, exception made for E308G where no changes are observed. Isothermal titration calorimetry (ITC) was used to characterize the interaction of the protein with NADP+ in terms of dissociation constant (K d), enthalpy (ΔH) and entropy (ΔS). K d values of 1.6 and 0.7 μM, ΔH of −13.9 Kcal/mol and −16.8 Kcal/mol, ΔS of −20.5 cal/mol/deg, and −28.5 cal/mol/deg were found for V257M and E158K respectively. E308G was found to be unable to bind the NADP+ cofactor, a result that is in line with the T m results. Circular dichroism also confirmed an overall lower stability of E308G, while NADP+ was found to give a strong positive shift of the T m stabilizing the structure of E158K (46.2 to 50.6 °C). Previous data highlighted significant differences in terms of activity among the SNPs of hFMO3. In this work a minor impact of the SNPs was found on the stability of the enzyme in the ligand free form, except for E308G, whereas the binding of NADP+ reveals major differences among WT and polymorphic variants that are all measurable in terms of heat capacity, enthalpy and secondary structure content. These data provide the first direct evidence of ligand stabilization effects on hFMO3 that can explain the differences observed in catalytic efficiencies and serve as the starting point for the development of inhibitors of this enzyme. [ABSTRACT FROM AUTHOR]

• Intact therapeutic mAbs analyzed by CZE- mass spectrometry under native conditions. • Simultaneous detection of folded, unfolded or dimer forms in a single analysis. • Involvement of Fab region in dimer formation was demonstrated by CZE-MS. Therapeutic monoclonal antibodies (mAbs) are complex glycoproteins and ensuring their safety, efficacy and quality is still challenging. Indeed, during their manufacturing process, they are exposed to several stresses that can lead to their denaturation, misfolding or dimerization. We report here a new method based on capillary electrophoresis coupled to native mass spectrometry (MS) with a sheath liquid interface to analyze an intact therapeutic mAb, Infliximab, under non-denaturing conditions that preserve its conformational heterogeneity as well as self-association without inducing further unfolding / denaturation. For capillary zone electrophoresis (CZE) separation, a triple layer coating using polybrene-dextran sulfate-polybrene was employed. A sheath liquid composed of isopropanol - water - acetic acid with a flow rate of 10 μL min−1 and mild MS conditions allowed optimal signal intensities. A specific mass spectrum was obtained for each Infliximab conformation in a "stressed" formulated preparation. This is the first time that within a single analysis different conformational states, i.e. native and unfolded monomers as well as dimers are simultaneously detected. The results and the lack of analytical bias arising from the CZE-MS conditions were confirmed by using atomic force microscopy (AFM) as an orthogonal technique. A middle-up approach combined to CZE-MS analysis of the stressed samples suggested that the dimer formation involved mostly Fab-Fab interactions. [ABSTRACT FROM AUTHOR]

[Display omitted] • Functionalized cationic gemini (C12-E2O2-C12) and single-chain surfactant (DTAC) instigated the myoglobin unfolding. • Beta -cyclodextrin (β -CD) induced the refolding of the unfolded Mb via formation of inclusion complex. • Fluorescence and UV–vis studies unveil nature and extent of binding between cationics and Mb. • Refolding of unfolded Mb using β -CD is found to be a reversible process. Herein, by pursuing consilience among several methodologies (different spectroscopic, molecular docking, and density functional theory (DFT)), we report that surfactants (functionalized cationic gemini, C 12 -E2O2-C 12 , and single-chain surfactant, DTAC) instigated myoglobin (Mb) unfolding and subsequently beta -cyclodextrin (β- CD) induced refolding of this unfolded Mb via inclusion complex formation. Fluorescence and UV–vis results unveil weak binding of the cationics at low concentrations followed by stronger interaction at higher concentrations before reaching the saturation point. Furthermore, refolding of the unfolded Mb using β -CD appears to be a reversible process. However, Mb does not completely recover its structure on refolding. From far-UV circular dichroism (CD) analysis, it is apparent that the α -helical content of Mb decreases/increases during unfolding/refolding process, which is further corroborated by Fourier transform infrared spectroscopy (FT-IR) results. Furthermore, fluctuations in the tertiary structure of Mb were detected through near-UV CD. Synchronous fluorescence together with molecular docking unravels the greater contribution of Trp in the involved interactions. DFT also substantiated the abstraction of C 12 -E2O2-C 12 /DTAC by β -CD. Fluorescence, far-UV CD and docking confirm the existence of negligible interaction between Mb and β -CD. The susceptibility of unfolding/refolding of Mb is considerably higher in the case of gemini than DTAC. In light of these results, it is strongly suggested that this work may prove fruitful for protein renaturation studies, which may address the interrelated issues of the biotechnological sector for the production of effective/affordable folding aids that could also be used to treat diseases linked to genetically engineered cells, caused by protein misfolding/aggregation. [ABSTRACT FROM AUTHOR]

State functions (e.g., ΔG) are path independent and quantitatively describe the equilibrium states of a thermodynamic system. Isothermal chemical denaturation (ICD) is often used to extrapolate state function parameters for protein unfolding in native buffer conditions. The approach is prudent when the unfolding/refolding processes are path independent and reversible, but may lead to erroneous results if the processes are not reversible. The reversibility was demonstrated in several early studies for smaller proteins, but was assumed in some reports for large proteins with complex structures. In this work, the unfolding/refolding of several proteins were systematically studied using an automated ICD instrument. It is shown that: (i) the apparent unfolding mechanism and conformational stability of large proteins can be denaturant-dependent, (ii) equilibration times for large proteins are non-trivial and may introduce significant error into calculations of ΔG, (iii) fluorescence emission spectroscopy may not correspond to other methods, such as circular dichroism, when used to measure protein unfolding, and (iv) irreversible unfolding and hysteresis can occur in the absence of aggregation. These results suggest that thorough confirmation of the state functions by, for example, performing refolding experiments or using additional denaturants, is needed when quantitatively studying the thermodynamics of protein unfolding using ICD. [ABSTRACT FROM AUTHOR]

Calreticulin of Brugia malayi (BmCRT) play very important role in host-parasite interaction. In previous study it was found that BmCRT is responsible for prevention of host classical complement pathway activation via its interaction with first component C1q of the human host. Therefore, BmCRT is an essential protein for parasite survival and an important drug target to fend filariasis. In the present study, we have carried out a systamatic biophysical characterization of BmCRT protein. Unfolding of BmCRT was found to be non-cooperative two-state process in the presence of both denaturant GdmCl and urea. The results also illustrated that protein lost its 50% activity at 1.5 M GdmCl and 3 M Urea. Partially unfolded and molten-globule like intermediate state was observed at 0.8 to 1.2 M GdmCl while Urea unfolding showed intermediate state at 1.2 to 1.6 M. Unfolding pathway monitored with the help of apolar quencher, favor above observations. All of these findings support the presence of detectable intermediate state during unfolding pathway of BmCRT. Furthermore, this study indicates that BmCRT is more stable toward temperature (Tm = 65 °C), pH and trypsin digestion. These differences in properties as compared to host can be fruitfully utilized for synthesis of compounds effective against the parasite. [ABSTRACT FROM AUTHOR]

The mutant His-107-Tyr of human carbonic anhydrase II (HCA II) is highly unstable and has long been linked to a misfolding disease known as carbonic anhydrase deficiency syndrome (CADS). High temperature unfolding trajectories of the mutant are obtained from classical molecular dynamics simulations and analyzed in a multi-dimensional property space. When projected along a reaction coordinate these trajectories yield four distinguishable sets of structures that map qualitatively to folding intermediates of this mutant postulated earlier from experiments. We present in this article a detailed analysis of representative structures and proton transfer activity of these intermediates. It is also suggested that under suitable experimental conditions, these intermediates may be distinguished using circular dichroism (CD) spectroscopy. Graphical Abstract. : We present a novel computational methodology of extracting representative structures of putative unfolding intermediates of a large protein from high temperature classical MD simulations. The extracted structures are investigated to assess their aggregation propensity and projected catalytic activity. [ABSTRACT FROM AUTHOR]

Guanylate kinase is one of the key enzymes in nucleotide biosynthesis. The study highlights the structural and functional properties of Brugia malayi Guanylate kinase (BmGK) in the presence of chemical denaturants. An inactive, partially unfolded, dimeric intermediate was observed at 1–2 M urea while GdnCl unfolding showed monomer molten globule like intermediate at 0.8–1.0 M. The results also illustrate the protective role of substrates in maintaining the integrity of the enzyme. The thermo stability of protein was found to be significantly enhanced in the presence of the substrates. Furthermore, binding of the substrates, GMP and ATP to BmGK changed its GdnCl induced unfolding pattern. Docking and molecular dynamic simulation performed for native BmGK, BmGK bound to GMP and GMP + ATP showed change in the fluctuation in the region between 130 and 150 residues. Arg134 lost its interaction with GMP and Arg145 interaction shifted to ATP after 40 ns simulation upon binding of ATP to BmGK-GMP complex. We, thus, propose the importance of specific rearrangements contributed by binding of substrates which participate in the overall stability of the protein. The work here emphasizes on detailed biophysical characterization of BmGK along with the significant role of substrates in modulating the structural and functional properties of BmGK. [ABSTRACT FROM AUTHOR]

• Two peak elution behavior is influenced by different parameters. • Temperature has a major role in on-column unfolding and aggregation. • Excipients have pH dependent effects in two peak elution behavior. • Some effects can be correlated to in-solution unfolding behavior using nanoDSF. Aggregation of proteins is a critical quality attribute and a major concern during the purification of therapeutic proteins, like monoclonal antibodies. In-solution experiments applying different stress scenarios, e.g., mechanical, or physical stresses, can determine the overall conformational stability of the protein to enhance drug product shelf-life. Several groups have reported surface-induced unfolding and aggregation of monoclonal antibodies and their derivatives during cation exchange chromatography, which results in a two-peak elution behavior of the protein and its species. We have investigated universal influencing factors, like temperature and hold time, on this phenomenon. The formation of the second peak is a kinetic process, which is strongly influenced by temperature during the hold time. However, our main focus was the application of excipients and their influence on the two-peak elution behavior. We compared the on-column screening results with results obtained through a "traditional" in-solution screening using nanoDSF. Mostly, stabilizing excipients, like Sucrose, show their stabilizing abilities in both systems, but some discrepancies, e.g., using Arginine, between the two orthogonal techniques show the potential of the on-column screening system to lead to unexpected results, which would not necessarily be visible in in-solution experiments. [ABSTRACT FROM AUTHOR]

Papillomavirus E2 protein that performs essential functions such as viral oncogene expression and replication represents specific target for therapeutic intervention. DNA-binding activity is associated with its C-terminal DNA-binding domain (DBD), while the N-terminal transactivation domain (TAD) is responsible for replication and transactivation functions. Although both demonstrate large dependence on dimerization for mediating their functions, K D for N-terminal dimerization is significantly high suggesting more dynamic role of this domain. However, unlike DBD, very little information is available on TAD dimerization, its folding and stability. Therefore, with an aim at delineating the regulatory switch of its dimerization, we have characterized high-risk HPV18 E2 TAD. Our studies demonstrate that E2 TAD is a weak but thermodynamically stable dimer ( K D ∼ 1.8 μM, Δ G H 2 O = 18.8 kcal mol −1 ) with α2–α3 helices forming the interface. It follows a three-state folding pathway, in which unfolding involves dissociation of a dimeric intermediate. Interestingly, 90% of the conformational free energy is associated with dimer dissociation (16.9 of 18.8 kcal mol −1 ) suggesting dimerization significantly contributes to its overall thermodynamic stability. These revelations might be important toward designing inhibitors for targeting dimerization or folding intermediates and hence multiple functions that E2 performs. [ABSTRACT FROM AUTHOR]

Urea interacts with the Na,K-ATPase, leading to reversible as well as irreversible inhibition of the hydrolytic activity. The enzyme purified from shark rectal glands is more sensitive to urea than Na,K-ATPase purified from pig kidney. An immediate and reversible inhibition under steady-state conditions of hydrolytic activity at 37 °C is demonstrated for the three reactions studied: the overall Na,K-ATPase activity, the Na-ATPase activity observed in the absence of K + as well as the K + -dependent phosphatase reaction (K-pNPPase) seen in the absence of Na + . Half-maximal inhibition is seen with about 1 M urea for shark enzyme and about 2 M urea for pig enzyme. In the presence of substrates there is also an irreversible inhibition in addition to the reversible process, and we show that ATP protects against the irreversible inhibition for both the Na,K-ATPase and Na-ATPase reaction, whereas the substrate paranitrophenylphosphate leads to a slight increase in the rate of irreversible inhibition of the K-pNPPase. The rate of the irreversible inactivation in the absence of substrates is much more rapid for shark enzyme than for pig enzyme. The larger number of potentially urea-sensitive hydrogen bonds in shark enzyme compared to pig enzyme suggests that interference with the extensive hydrogen bonding network might account for the higher urea sensitivity of shark enzyme. The reversible inactivation is interpreted in terms of domain interactions and domain accessibilities using as templates the available crystal structures of Na,K-ATPase. It is suggested that a few interdomain hydrogen bonds are those mainly affected by urea during reversible inactivation. [ABSTRACT FROM AUTHOR]

The unfolding of the reduced human serum albumin (HSA) was simulated by generating four molecular dynamics (MD) trajectories of 160 ns each at 350, 375, 400, and 425 K, respectively. A principal components analysis (PCA) was performed on the four trajectories. Based on this analysis, 17 representative protein conformers were identified and subsequently used to construct a sequence of partially unfolded structures. They were ordered according to their decreasing α-helix fractions. The structural evolution in this unfolding sequence was found to be continuous at global but also at local level supporting the hypothesis that the protein unfolding pathway is not significantly dependent on the simulation temperature. As a result, the α-helix fraction of the protein appears to be a good reaction coordinate for the unfolding process, as it was previously suggested by experiments. Based on this observation, two conformers in the unfolding sequence were predicted to be close to the equilibrium structure of reduced HSA thus providing a first theoretical model for this protein form. [ABSTRACT FROM AUTHOR]

Defensins are a class of immune peptides with a broad range of activities against bacterial, fungal and viral pathogens. Besides exerting direct anti-microbial activity via dis-organization of bacterial membranes, defensins are also able to neutralize various unrelated bacterial toxins. Recently, we have demonstrated that in the case of human a- and ß-defensins, this later ability is achieved through exploiting toxins' marginal thermodynamic stability, i.e. defensins act as molecular anti-chaperones unfolding toxin molecules and exposing their hydrophobic regions and thus promoting toxin precipitation and inactivation [Kudryashova et al. (2014) Immunity 41, 709-721]. Retrocyclins (RCs) are humanized synthetic ?-defensin peptides that possess unique cyclic structure, differentiating them from a- and ß-defensins. Importantly, RCs are more potent against some bacterial and viral pathogens and more stable than their linear counterparts. However, the mechanism of bacterial toxin inactivation by RCs is not known. In the present study, we demonstrate that RCs facilitate unfolding of bacterial toxins. Using differential scanning fluorimetry (DSF), limited proteolysis and collisional quenching of internal tryptophan fluorescence, we show that hydrophobic regions of toxins normally buried in the molecule interior become more exposed to solvents and accessible to proteolytic cleavage in the presence of RCs. The RC-induced unfolding of toxins led to their precipitation and abrogated activity. Toxin inactivation by RCs was strongly diminished under reducing conditions, but preserved at physiological salt and serum concentrations. Therefore, despite significant structural diversity, a-, ß- and ?-defensins employ similar mechanisms of toxin inactivation, whichmay be shared by anti-microbial peptides from other families. [ABSTRACT FROM AUTHOR]

• The two-peak elution behavior of a mAb in CEX has many influencing factors. • The mAb shows surface-induced unfolding, reversible association, and aggregation. • pH value, salt concentration and ligand density play a significant role. • The ligand density effect correlates with the lower resin pore pH during hold times. • Low resin pH values destabilize and unfold the protein and influences the binding. Cation exchange chromatography, as part of the monoclonal antibody purification train, is known as a mild polishing technique. However, in the last couple of years, more and more publications have shown unusual elution behavior, resulting from e.g. on-column (reversible) unfolding and aggregation of the predominantly mAb molecules. The stability of the investigated protein seems to play a significant role in this phenomenon. We have used a glycosylated IgG1 antibody as a model protein and investigated several influencing factors, including pH value and ligand density variations of three prototype Fractogel® cation exchange resins. Ligand density, pH and salt concentration are the main contributing factors in the Donnan effect, i.e. distribution of ions, between resin pore volume and bulk volume. This leads to a significantly lower pH value the protein is subjected to during the on-column hold time and therefore influences the conformational stability of our protein. Nano-DSF and kinetic SEC measurements show that the protein is destabilized at low pH values, but also, that the binding to the CEX resin and the elution with increasing salt concentration is responsible for the resulting two-peak elution behavior and partially reversible unfolding and aggregation. [ABSTRACT FROM AUTHOR]

Differential scanning calorimetry (DSC) was applied to investigate the effect of cholesterol on the thermotropic properties of the lipid membrane (DPPC and DPPE). The thermostability and unfolding of solubilized and reconstituted Na,K-ATPase in DPPC:DPPE:cholesterol-liposomes was also studied to gain insight into the role of cholesterol in the Na,K-ATPase modulation of enzyme function and activity. The tertiary system (DPPC:DPPE:cholesterol) (molar ratio DPPC:DPPE equal 1:1) when cholesterol content was increased from 0% up to 40% results in a slight decrease in the temperature of transition and enthalpy, and an increase in width. We observed that, without heating treatment, at 37 °C, the activity was higher for 20 mol% cholesterol. However, thermal inactivation experiments showed that the enzyme activity loss time depends on the cholesterol membrane content. The unfolding of the enzyme incorporated to liposomes of DPPC:DPPE (1:1 mol) with different cholesterol contents, ranging from 0% to 40% mol was also studied by DSC. Some differences between the thermograms indicate that the presence of lipids promotes a conformational change in protein structure and this change is enough to change the way Na,K-ATPase thermally unfolds. [ABSTRACT FROM AUTHOR]

A glycosylated IgG2 monoclonal antibody exhibits a two-peak elution behavior when loaded on a strong cation exchange column and eluted with either a linear salt gradient or two salt steps at increasing salt concentrations. The two-peak behavior is more pronounced for conditions where the initial antibody binding is stronger, i.e. at lower pH and buffer concentration, where the hold time prior to elution is longer, where the protein mass load is lower, and where the load flow rate is higher. The effect is also dependent on the resin type, being prominent for the polymer-functionalized resin Fractogel EMD SO3- and virtually absent for a macroporous resin with similar backbone but no grafted polymers. Size exclusion chromatography and dynamic light scattering show that the early eluting peak consists exclusively of the native monomeric species while the late eluting peak is a mixture of monomeric and aggregated species. Batch adsorption/desorption experiments show that the bound protein can be desorbed in two steps, with a fraction desorbed in 0.33M NaCl, corresponding to native monomer, and a second fraction desorbed in 1M NaCl. The latter fraction decreases with protein mass load and becomes almost negligible when the resin is initially completely saturated with protein. Confocal laser scanning microscopy showed that the two-peak elution/desorption behavior is related to the unique kinetics of protein binding in the Fractogel resin. Following partial loading of the resin, the bound protein migrates toward the center of the particles during a hold step and is redistributed across the particle volume attaining low local bound protein concentrations. For these conditions the protein is apparently destabilized forming a strongly-bound unfolded intermediate that, in turn, generates aggregates upon elution in high salt. [ABSTRACT FROM AUTHOR]

Hydrogen-deuterium exchange mass spectrometry (HX-MS) with proteolytic fragmentation is used to determine patterns of unfolding, as measured by increased solvent exposure, with peptide-level resolution for a glycosylated monoclonal antibody both when eluted from a tentacle-type cation exchange column (Fractogel EMD SO3-) and while bound to the resin. Two peaks are obtained when the bound protein is eluted with either a NaCl gradient or with two NaCl steps at increasing concentration. The first, early eluting peak contains only monomeric species whose structure is consistent with the native monomer. The second, late eluting peak contains a mixture of monomeric and aggregated species. The monomeric species in this mixture is also found to have a structure consistent with that of the native mAb, showing no evidence of increased solvent exposure. The aggregated species show instead significant unfolding in areas of the protein structure contained within the Fc region. The same peptides that exhibit the greatest level of solvent exposure in the aggregated species are also found in the fraction of protein that elutes from the resin only at high salt concentration, indicating that the aggregates are formed when the strongly-bound unfolded intermediate is desorbed at high salt. There is no evidence that the unfolded intermediate, formed while the protein is bound on the resin, is present in any of the eluted fractions indicating that, upon desorption from the resin, the intermediate either quickly refolds or forms aggregates which end-up co-eluting with the refolded protein at high salt concentrations. [ABSTRACT FROM AUTHOR]

Abstract: The regulation of enzymatic activity and unfolding studies of arginine kinase (AK) from various invertebrates have been the focus of investigation. To gain insight into the structural and folding mechanisms of AK from Euphausia superba (ESAK), we purified ESAK from muscle properly. The enzyme behaved as a monomeric protein with a molecular mass of about 40kDa and had pH and temperature optima of 8.0 and 30°C, respectively. The and for the synthesis of phosphoarginine were 0.30 and 0.47mM, respectively, and was 282.7s−1/mM. A study of the inhibition kinetics of structural unfolding in the denaturant sodium dodecyl sulfate (SDS) was conducted. The results showed that ESAK was almost completely inactivated by 1.0mM SDS. The kinetics analyzed via time-interval measurements revealed that the inactivation was a first-order reaction, with the kinetic processes shifting from a monophase to biphase as SDS concentrations increased. Measurements of intrinsic and 1-anilinonaphthalene-8-sulfonate-binding fluorescence showed that SDS concentrations lower than 5mM did not induce conspicuous changes in tertiary structures, while higher concentrations of SDS exposed hydrophobic surfaces and induced conformational changes. These results confirmed that the active region of AK is more flexible than the overall enzyme molecule. [Copyright &y& Elsevier]

Highlights: [•] Arginine kinase (AK) folding mechanisms in the presence of Zn2+. [•] Zn2+-induced aggregation phenomenon of AK from the Antarctic krill (Euphausia superba). [•] Comparisons between conformational changes and catalytic functions of AK with Zn2+ binding. [•] Several osmolytes roles for preventing AK aggregation. [ABSTRACT FROM AUTHOR]

In most beetles, the hind wings are thin and fragile; when at rest, they are held over the back of the beetle. When the hind wing unfolds, it provides the necessary aerodynamic forces for flight. In this paper, we investigate the hydraulic mechanism of the unfolding process of the hind wings in Dorcus titanus platymelus (Oder: Coleoptera). The wing unfolding process of Dorcus titanus platymelus was examined using high speed camera sequences (400 frames/s), and the hydraulic pressure in the veins was measured with a biological pressure sensor and dynamic signal acquisition and analysis (DSA) during the expansion process. We found that the total time for the release of hydraulic pressure during wing folding is longer than the time required for unfolding. The pressure is proportional to the length of the wings and the body mass of the beetle. A retinal camera was used to investigate the fluid direction. We found that the peak pressures correspond to two main cross-folding joint expansions in the hind wing. These observations strongly suggest that blood pressure facilitates the extension of hind wings during unfolding. [ABSTRACT FROM AUTHOR]

Abstract: The adsorption of DNAs in G-quadruplex solution onto 13nm gold nanoparticles (AuNPs) was studied through monitoring of the localized surface plasmon resonance (LSPR) absorbance of 13nm AuNPs at 520 and 650nm (A 650/A 520) in the solutions of three widely studied guanine-rich sequences, TBA(5′-GGTTGGTGTGGTTGG-3′), PW17(5′-GGGTAGGGCGGGTTGGG-3′), and PSO (5′-GGGTTAGGGTTAGGGTTAGGG-3′). It was found that the degree of adsorption of DNAs in Pb2+ stabilized G-quadruplex (G-Pb2+) solutions is up to 93% after more than 5h of incubation. Furthermore, the lead concentrations in the solutions containing G-quadruplex and AuNP were analyzed by an inductively coupled plasma atomic emission spectrometer. The results showed that Pb2+ had been released from the G-quadruplexes, which means the G-quadruplexes may be unfolded in the presence of AuNP. This interaction between G-quadruplexes and AuNP demonstrated that long time incubation between DNAs and AuNPs would possibly make it unable to distinguish G-quadruplex from ssDNA. Thus, a biosensing system consisting of PW17 and AuNPs was developed to detect Pb2+. It was found that the LSPR responses at A 650/A 520 were sensitive to [Pb2+]. However, the sensitivity of the system was interfered by the potential unfolding of PW17-Pb2+ in the presence of AuNPs. This unexpected adverse effect of AuNPs on DNA-based biosensors should be taken into consideration in the future development of biosensing systems that are based on ssDNA aptamers and unmodified AuNPs. [Copyright &y& Elsevier]

Highlights: [•] Earliest report on the conformational transition studies of L1-CCRH1 from Leucaena leucocephala. [•] Characterization of acid induced molten globule. [•] Rapid structural rearrangement and thermal aggregation at and above 50°C. [•] Different response of the molten globule towards thermal and chemical denaturation as compared to native protein. [ABSTRACT FROM AUTHOR]

The effects of 3 hours’ exposure to mobile phone microwaves at 1765 MHz at the power density around 800 mW/m2on the secondary structure of myoglobin in D2O solution were investigated by Fourier transform infrared spectroscopy. A significant shift to lower frequencies of the amide I vibration was observed after exposure. Furthermore, a significant increasing of the β-sheet components with respect to the α-helix content after exposure was highlighted after applying Fourier self-deconvolution analysis in the amide I region. These results led to the conclusion that mobile phone microwaves induce unfolding of myoglobin and formation of aggregates. [ABSTRACT FROM AUTHOR]

To elucidate the structural stability and the unfolding dynamics of the animal prion protein, the temperature induced structural evolution of turtle prion protein (tPrPc) and bank vole prion protein (bvPrPc) have been performed with molecular dynamics (MD) simulation. The unfolding behaviors of secondary structures showed that the α-helix was more stable than β-sheet. Extension and disruption of β-sheet commonly appeared in the temperature induced unfolding process. The conversion of α-helix to π-helix occurred more readily at the elevating temperature. Furthermore, it was suggested in this work that the unfolding of prion protein could be regulated by the temperature. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

There are over 100 mutations in Cu/Zn superoxide dismutase (SOD1) that result in a subset of familial amyotrophic lateral sclerosis (fALS) cases. The hypothesis that dissociation of the dimer, misfolding of the monomer and subsequent aggregation of mutant SOD1 leads to fALS has been gaining support as an explanation for how these disparate missense mutations cause the same disease. These forms are only responsible for a fraction of the ALS cases; however, the rest are sporadic. Starting with a folded apo monomer, the species considered most likely to be involved in misfolding, we used high-temperature all-atom molecular dynamics simulations to explore the events of the wild-type protein unfolding through the denatured state. All simulations showed early loss of structure along the β5–β6 edge of the β-sandwich, supporting earlier findings of instability in this region. Transition state structures identified from the simulations are in good agreement with experiment, providing detailed, validated molecular models for this elusive state. Furthermore, we compare the process of thermal unfolding investigated here to that of the lethal A4V mutant-induced unfolding at physiological temperature and find that the pathways are very similar. [ABSTRACT FROM AUTHOR]

The detailed structure and dynamics of the chromatin fibre and their relation to gene regulation represent important open biological questions. Recent advances in single-molecule force spectroscopy experiments have addressed these questions by directly measuring the forces that stabilize and alter the folded states of chromatin, and by investigating the mechanisms of fibre unfolding. We present examples that demonstrate how complementary modelling approaches have helped not only to interpret the experimental findings, but also to advance our knowledge of force-induced events such as unfolding of chromatin with dynamically bound linker histones and nucleosome unwrapping. [ABSTRACT FROM AUTHOR]

Abstract: During the last two decades single-molecule manipulation techniques such as atomic force microscopy (AFM) has risen to prominence through their unique capacity to provide fundamental information on the structure and function of biomolecules. Here we describe the use of single-molecule AFM to track protein unfolding and refolding pathways, enzymatic catalysis and the effects of osmolytes and chaperones on protein stability and folding. We will outline the principles of operation for two different AFM pulling techniques: length clamp and force-clamp and discuss prominent applications. We provide protocols for the construction of polyproteins which are amenable for AFM experiments, the preparation of different coverslips, choice and calibration of AFM cantilevers. We also discuss the selection criteria for AFM recordings, the calibration of AFM cantilevers, protein sample preparations and analysis of the obtained data. [Copyright &y& Elsevier]

Abstract: Unfolding by chemical denaturants and the linear extrapolation method are widely used to determine the free energy of proteins. Ribonuclease 3 from bullfrog shows an extraordinary behavior in guanidinium hydrochloride in comparison to its homologues ribonuclease A and onconase with a high transition midpoint of denaturation but an apparently low cooperativity. The analysis of the interdependence of thermal, urea-, and guanidine hydrochloride-induced unfolding revealed that whereas addition of urea resulted in the expected destabilization of all three proteins, guanidine hydrochloride acted diversely: in contrast to ribonuclease A and onconase, both of which were destabilized as expected, low concentrations of guanidine hydrochloride significantly stabilize ribonuclease 3 from bullfrog. This stabilizing effect was endorsed by in silico docking studies. [Copyright &y& Elsevier]

Abstract: An electrochemical approach based on protein controllable unfolding was developed and applied in combination with in-situ reaction in order to investigate the electrocatalytic pathway for hemoglobin (Hb) toward nitric oxide (NO). Hb was entrapped in a dimethyldidodecylammonium bromide (DDAB) film modified glassy carbon electrode (DDAB/Hb/GCE). Two typical denaturants of acid and urea were synergistically utilized to control the incorporated Hb to a most unfolded state without losing heme groups. Under optimal conditions, the unfolded DDAB/Hb/GCE exhibited accelerated direct electron transfer. The sensitivities for the detection of ascorbic acid (AA), NaNO2 and NO were improved as 3, 10 and 12 times higher than those on the native DDAB/Hb/GCE, and the limits of detection (LODs) for AA, NaNO2 and NO were down to 0.33, 0.83 and 0.063μM, respectively. The unfolded DDAB/Hb/GCE was further applied for the investigation of Hb–NO interaction in NaNO2 solution. With successive additions of AA, NO was generated in situ on DDAB/Hb/GCE. A new reduction peak of the intermediate HbFe(II)–HN2O2 was successfully revealed near −0.65V. The whole electrocatalytic mechanism was proposed and verified by density functional theory. The method can be a promising platform for facile study of the interaction between NO and heme proteins. [Copyright &y& Elsevier]

Abstract: Curcumin influences the transition point, the concentration of denaturant required to effect 50% of the total change, of myoglobin denaturation. Curcumin enhances absorbance of myoglobin at 280nm with a binding constant K =3.0×104 M−1 whereas fluorescence of curcumin is quenched by myoglobin with a Stern–Volmer association constant of 2.5×105 M−1. Unfolding process of myoglobin–curcumin induces a recovery in fluorescence lifetime loss. The gain in time-resolved fluorescence lifetime during unfolding has been again lost during refolding of curcumin–myoglobin complex by dilution process suggesting partial reversibility of unfolding process for both myoglobin and curcumin–myoglobin complex. [Copyright &y& Elsevier]

Abstract: The Escherichia coli RecA protein is a naturally aggregated protein complex that is affected by the presence of salts. In order to gain further insight into the nature of the ion-interactions on a naturally aggregating protein we used circular dichroism (CD), fluorescence and dynamic light scattering (DLS) to study the effects of different concentrations of MgCl2, CaCl2, NaCl, Na2SO4, and MgSO4 on RecA structure and thermal unfolding. The results show unique ion influences on RecA structure, aggregation, unfolding transitions and stability and the anion effects correlate with the reverse Hofmeister series. The mechanisms of the ion-induced changes most likely result from specific ion binding, changes in the interfacial tension and altered protein–solvent interactions that may be especially important for protein–protein interactions in naturally aggregating proteins. The presence of some ions leads to the formation of RecA complexes that are resistant to complete denaturation and nonspecific aggregation. [Copyright &y& Elsevier]

Abstract: Superoxide dismutases are enzymes that protect biological systems against oxidative damage caused by superoxide radicals. In this paper, a detailed characterization is presented on the stability of SmSOD, the dimeric cambialistic superoxide dismutase from the dental pathogenic microorganism Streptococcus mutans, towards temperature and guanidine hydrochloride. Thermal and chemical denaturations were investigated by means of circular dichroism, fourth-derivative UV spectroscopy and fluorescence measurements. Data indicate that SmSOD is endowed with a significant thermostability and that both its thermal and guanidine hydrochloride-induced unfolding processes occur through a three-state model, characterized by a catalytically active dimeric intermediate species. To our knowledge, SmSOD is the smallest known dimeric protein that populates a well-structured active dimeric rather than a monomeric intermediate during unfolding processes. [Copyright &y& Elsevier]

Periplasmic metal binding protein characterized by high histidine content was cloned from moderate halophile, Chromohalobacter salexigens. The protein, termed histidine-rich metal binding protein (HP), was expressed in and purified from E. coli as a native form. HP bound to Ni- and Cu-loaded chelate columns with high affinity, and Co- and Zn-columns with moderate affinity. Although the secondary structure was not grossly altered by the addition of 0.2-2.0 M NaCl, the thermal transition pattern was considerably shifted to higher temperature with increasing salt concentration: melting temperature was raised by ~20 °C at 2.0 M NaCl over the melting temperature at 0.2 M NaCl. HP showed reversible refolding from thermal melting in 0.2-1.15 M NaCl, while it formed irreversible aggregates upon thermal melting at 2 M NaCl. Addition of 0.01-0.1 mM NiSO stabilized HP against thermal melting with high reversibility, while addition above 0.5 mM resulted in irreversible melting due to aggregation. [ABSTRACT FROM AUTHOR]

Abstract: The unfolding of hen egg-white lysozyme dissolved both in D2O and CH3CH2OD/D2O was studied by Fourier Transform Infrared (FTIR) absorption spectroscopy at different protein concentrations. A detailed description of the local and global rearrangement of the secondary structure upon a temperature increase, in the range 295 to 365K, was obtained through the analysis of the amide I band. Thermodynamic parameters for the melting, and the effect of the co-solvent in determining a change in thermal stability of the protein were evaluated. The protein-protein interactions were also followed as a function of temperature: a strong dependence of the cluster stability and aggregation yield on the solvent composition was observed. Finally, FTIR spectra taken at successive time steps of the aggregation enabled intermolecular contacts to be monitored as a function of time, and kinetic information to be obtained showing that both unfolded and folded states of lysozyme act as reactants for the clustering event. [Copyright &y& Elsevier]

Equilibrium unfolding of A. niger RNase with chemical denaturants, for example GuHCl and urea, and thermal unfolding have been studied as a function of pH using fluorescence, far-UV, near-UV, and absorbance spectroscopy. Because of their ability to affect electrostatic interactions, pH and chemical denaturants have a marked effect on the stability, structure, and function of many globular proteins. ANS binding studies have been conducted to enable understanding of the folding mechanism of the protein in the presence of the denaturants. Spectroscopic studies by absorbance, fluorescence, and circular dichroism and use of K2D software revealed that the enzyme has α + β type secondary structure with approximately 29% α-helix, 24% β-sheet, and 47% random coil. Under neutral conditions the enzyme is stable in urea whereas GuHCl-induced equilibrium unfolding was cooperative. A. niger RNase has little ANS binding even under neutral conditions. Multiple intermediates were populated during the pH-induced unfolding of A. niger RNase. Urea and temperature-induced unfolding of A. niger RNase into the molten globule-like state is non-cooperative, in contrast to the cooperativity seen with the native protein, suggesting the presence of two parts/domains, in the molecular structure of A. niger RNase, with different stability that unfolds in steps. Interestingly, the GuHCl-induced unfolding of the A state (molten globule state) of A. niger RNase is unique, because a low concentration of denaturant not only induces structural change but also facilitates transition from one molten globule like state (A) into another (I). [ABSTRACT FROM AUTHOR]

Abstract: We have used fluorescence spectroscopy techniques such as fluorescence correlation spectroscopy and fluorescence anisotropy decay on a wide time range, from nanoseconds to seconds, to investigate the unfolding kinetics induced by guanidinium chloride of GFPMut2 and its point mutation H148G, which has proved to be relevant for GFP photochemistry and photophysics. The mutation affects the unfolding kinetics of GFP leading to a much faster process at alkaline pH values, where protonation dynamics is negligible, that can be ascribed to a twofold role of His148, either as a proton shutter towards the chromophore and as a conformation stabiliser. For both mutants a soft region located near beta-strand 3 is found that starts to gain flexibility in the ns range at denaturant concentrations far lower than those required to turn off the chromophore fluorescence, as derived from the anisotropy decay of an extrinsic probe covalently bound to the proteins. [Copyright &y& Elsevier]

Abstract: The electron transfer (ET) process of progressively unfolded bovine cytochrome c immobilized on different self-assembled monolayers (SAMs) was investigated. Insight is gained on the role of the SAM surface on the functionality of the partially unfolded and non-native forms of the adsorbed protein. Direct electrochemical measurements were performed on cytochrome c adsorbed on mercaptopyridine (MP) and mixed 11-mercapto-1-undecanoic acid/11-mercapto-1-undecanol (MUA/MU) at varying temperature, in the presence of urea as unfolding agent. Under strongly unfolding conditions, a non-native form of cytochrome c, in which the methionine ligand is replaced by a histidine, was observed on both MP and MUA/MU SAMs. The E°′ of the native form, in which the haem is axially coordinated by methionine and histidine, slightly shifts to negative values upon increasing urea concentration. However, the non-native bis-histidinate species shows a much lower E°′ value (by approximately 0.4V) which is by far enthalpic in origin and largely determined by axial ligand swapping. Analysis of the reduction enthalpies and entropies and of the ET rate constants indicate that the nature of the SAM (hydrophilic or anionic) results in changes in the conformational rearrangement of the cytochrome c under unfolding conditions. [Copyright &y& Elsevier]

Abstract: The cellular isoform of the prion protein PrPC is a Cu2 +-binding cell surface glycoprotein that, when misfolded, is responsible for a range of transmissible spongiform encephalopathies. As changes in PrPC conformation are intimately linked with disease pathogenesis, the effect of Cu2+ ions on the structure and stability of the protein has been investigated. Urea unfolding studies indicate that Cu2+ ions destabilise the native fold of PrPC. The midpoint of the unfolding transition is reduced by 0.73±0.07 M urea in the presence of 1 mol equiv of Cu2+. This equates to an appreciable difference in free energy of unfolding (2.02±0.05 kJ mol−1 at the midpoint of unfolding). We relate Cu2 +-induced changes in secondary structure for full-length PrP(23–231) to smaller Cu2 + binding fragments. In particular, Cu2+-induced structural changes can directly be attributed to Cu2+ binding to the octarepeat region of PrPC. Furthermore, a β-sheet-like transition that is observed when Cu ions are bound to the amyloidogenic fragment of PrP (residues 90–126) is due only to local Cu2+ coordination to the individual binding sites centred at His95 and His110. Cu2+ binding does not directly generate a β-sheet conformation within PrPC; however, Cu2+ ions do destabilise the native fold of PrPC and may make the transition to a misfolded state more favourable. [Copyright &y& Elsevier]

Abstract: Efficient immobilization of urease was achieved upon dioxane-induced unfolding/refolding strategy on alkyl modified porous silica with an average pore size of 66nm. Structural exploration of the urease was carried out to find the optimum condition of solvent polarity which provides efficient adsorptive immobilization through emphasizing on dual approaches: creation of hydrophobic intermediates as molten globule like states and improved accessibility of substituted alkyl chains. The optimum percent volume of dioxane in phosphate buffer to fulfill such aim was achieved at 30% (v/v). Restoring of native-like secondary structure was observed using circular dichroism; moreover, improved exposure of hydrophobic surfaces of urease was confirmed using a set of UV-analysis, intrinsic fluorescence, and differential 8-anilino-1-naphthalene-sulfonate fluorescence spectroscopy, at 30% as an optimum concentration of dioxane. The yield of immobilization was doubled using reversible denaturation approach and storage stability of the immobilization product of urease was noticeably improved (half live of the multi-used immobilized urease was resulted to be 6.5 folds higher than the half live of the free enzyme). Moreover, 7–13.5 folds activation of the enzyme was resulted upon immobilization. Improved immobilization ensures the efficiency of this strategy, for applied approaches, along with providing further evidences for enhanced surface hydrophobicity of the multimeric urease at defined concentration of dioxane. [Copyright &y& Elsevier]

Abstract: In the present study, the thermal unfolding of lysozyme in D2O and D2O/CH3CH2OD solutions is analyzed by means of FTIR spectroscopy. A detailed investigation of the conformational rearrangement of the protein upon heating is achieved by inspection of the amide bands. The effects of partial and total deuteration on amide groups are also investigated for they suggest important information about exposure to the solvent. Finally, a comparison between the characteristics of the unfolding process of lysozyme in water and in ethanol/water solutions is performed to clarify some misinterpretation about the possible formation of intermediates. [Copyright &y& Elsevier]

High-pressure Fourier-transform infrared (FTIR) spectroscopy was used to determine the pressure and temperature stability of Mal d1. This study was triggered by contradictory results in the literature regarding the success of pressure treatment in the destruction of the allergen. The protein unfolded at 55°C when heated at normal atmospheric pressure. We also studied the effect exerted on pressure stability by environmental factors, which can be important for the stability of the protein in the apple. The pressure unfolding was measured under different pD conditions, and the effect of sugar mixture similar to that of the apple and the effect of ionic strength were also studied. In all cases the allergen unfolded with a transition midpoint in the range of 150-250 MPa. Unfolding was irreversible and was followed by aggregation of the unfolded protein. Lowering the pD destabilized the protein, while addition of sugar mixture and of KCl had stabilizing effect. [ABSTRACT FROM AUTHOR]

Abstract: The activity and conformational changes of ficin (EC 3.4.22.3), a cysteine protease from Ficus carica have been investigated during the denaturation by urea and guanidine hydrochloride (GuHCl). The denaturation of ficin was followed by activity measurements, fluorescence and circular dichroism (CD) spectroscopic studies. The enzyme activity decreased significantly at low concentration of both urea and GuHCl before unfolding of the enzyme molecule. The enzyme molecule was resistant for unfolding by urea under neutral conditions even at higher concentrations. However, the protein is susceptible to unfolding by urea at lower pH and transition follows a cooperative two-state rule with increasing concentration of urea. On the other hand, ficin molecule loses its complete structure in presence of 4M GuHCl under neutral conditions. The GuHCl-induced unfolding occurs in a simple two-state cooperative process. These results indicate the differential structural stability and fragility of active site of the enzyme towards denaturation by urea and GuHCl. [Copyright &y& Elsevier]

Coagulation factor IX/coagulation factor X binding protein from the venom of Agkistrodon halys Pallas (AHP IX/X-bp) is a unique coagulation factor IX/coagulation factor X binding protein (IX/X-bp). Among all IX/X-bps identified, only AHP IX/X-bp is a Ca- and Zn-binding protein. The binding properties of Ca and Zn ions binding to apo-AHP IX/X-bp and their effects on the stability of the protein have been investigated by isothermal titration calorimetry, fluorescence spectroscopy, and differential scanning calorimetry. The results show that AHP IX/X-bp has two metal binding sites, one specific for Ca with lower affinity for Zn and one specific for Zn with lower affinity for Ca. The bindings of Ca and Zn in the two sites are entropy- and enthalpy-driven. The binding affinity of AHP IX/X-bp for Zn is 1 order of magnitude higher than for Ca for either high-affinity binding or low-affinity binding, which accounts for the existence of one Zn in the purified AHP IX/X-bp. Guanidine hydrochloride (GdnHCl)-induced and thermally induced denaturations of Ca-Ca-AHP IX/X-bp, Zn-Zn-AHP IX/X-bp, and Ca-Zn-AHP IX/X-bp are all a two-state processes with no detectable intermediate state(s), indicating the Ca/Zn-induced tight packing of the protein. Ca and Zn increase the structural stability of AHP IX/X-bp against GdnHCl or thermal denaturation to a similar extent. Although Ca and Zn have no obvious effect on the secondary structure of AHP IX/X-bp, they induce different rearrangements in local conformation. The Zn-stabilized specific conformation of AHP IX/X-bp may be helpful to its recognition of the structure of coagulation factor IX. This work suggests that in vitro, Ca plays a structural rather than an active role in the anticoagulation of AHP IX/X-bp, whereas Zn plays both structural and active roles in the anticoagulation. In blood, Ca binds to AHP IX/X-bp and stabilizes its structure, whereas Zn cannot bind to AHP IX/X-bp owing to the low Zn concentration. AHP IX/X-bp prolongs the clotting time in vivo through its binding only with coagulation factor X/activated coagulation factor X. [ABSTRACT FROM AUTHOR]

Abstract: The relevance of partially ordered states of proteins (such as the molten-globule state) in cellular processes is beginning to be understood. We examined the conformational transitions in a multimeric and high molecular weight class II α-mannosidase from Canavalia ensiformis (Jack Bean) (Jbα-man) utilizing intrinsic fluorescence, solute quenching, hydrophobic dye binding, size exclusion chromatography and circular dichroism (CD) spectroscopy for the protein in presence of Guanidine hydrochloride (GdnHCl). The decomposition analysis of the protein spectra obtained during unfolding showed progressive appearance of class S, I, II and III trp. The parameter A and spectral center of mass showed multi state unfolding of the protein and phase diagram analysis revealed formation of an intermediate of Jbα-man in the vicinity of 1M GdnHCl. The intermediate exhibited compact secondary and distorted tertiary structure with exposed hydrophobic amino acids on the surface, indicating the molten-globule nature. The dissociation, partial unfolding and aggregation of Jbα-man occurred simultaneously during chemical denaturation. The molten-globule possessed slightly higher hydrodynamic radius, perturbance in the structure up to 60°C and stability of the structure up to 80°C unlike the native Jack Bean α-mannosidase. The modes of chemical and thermal denaturation of the native protein were different. The solute quenching parameters confirmed the altered confirmation of the intermediate. Taken together, our results constitute one of the early reports of formation of GdnHCl induced molten globule in a class II α-mannosidase. [Copyright &y& Elsevier]

Abstract: Neuronal Src (n-Src) is an alternative isoform of Src kinase containing a 6-amino acid insert in the SH3 domain that is highly expressed in neurons of the central nervous system (CNS). To investigate the function of n-Src, wild-type n-Src, constitutively active n-Src in which the C-tail tyrosine 535 was mutated to phenylalanine (n-Src/Y535F) and inactive n-Src in which the lysine 303 was mutated to arginine in addition to the mutation of Y535F (n-Src/K303R/Y535F), were expressed and purified from Escherichia coli BL21(DE3) cells. We found that all three types of n-Src constructs expressed at very high yields (∼500mg/L) at 37°C, but formed inclusion bodies. In the presence of 8M urea these proteins could be solubilized, purified under denaturing conditions, and subsequently refolded in the presence of arginine (0.5M). These Src proteins were enzymatically active except for the n-Src/K303R/Y535F mutant. n-Src proteins expressed at 18°C were soluble, albeit at lower yields (∼10–20mg/L). The lowest yields were for n-Src/Y535F (∼10mg/L) and the highest for n-Src/K303R/Y535F (∼20mg/L). We characterized the purified n-Src proteins expressed at 18°C. We found that altering n-Src enzyme activity either pharmacologically (e.g., application of ATP or a Src inhibitor) or genetically (mutation of Y535 or K303) was consistently associated with changes in n-Src stability: an increase in n-Src activity was coupled with a decrease in n-Src stability and vice versa. These findings, therefore, indicate that n-Src activity and stability are interdependent. Finally, the successful production of functionally active n-Src in this study indicates that the bacterial expression system may be a useful protein source in future investigations of n-Src regulation and function. [ABSTRACT FROM AUTHOR]