Loss Of Tertiary Structure Research Articles

The transported cations impose differences in the thermostability of the gastric H,K-ATPase. A kinetic analysis

This work analyses the thermostability of a membrane protein, the gastric H,K-ATPase, by means of a detailed kinetic characterization of its inactivation process, which showed to exhibit first-order kinetics. We observed parallel time courses for the decrease of ATPase activity, the decrease of the autophosphorylation capacity and the loss of tertiary structure at 49 °C. Higher temperatures were required to induce a significant change in secondary structure. The correspondence between the kinetics of Trp fluorescence measured at 49 °C and the decrease of the residual activity after heating at that temperature, proves the irreversibility of the inactivation process. Inactivation proceeds at different rates in E1 or E2 conformations. The K+-induced E2 state exhibits a lower inactivation rate; the specific effect is exerted with a K0.5 similar to that found at 25 °C, providing a further inkling that K+ occlusion by the H,K-ATPase is not really favoured. Increasing [H+] from pH 8 to pH 7, which possibly shifts the protein to E1, produces a subtle destabilizing effect on the H,K-ATPase. We performed a prediction of potential intramolecular interactions and found that the differential stability between E1 and E2 may be mainly explained by the higher number of hydrophobic interactions in the α- and β-subunits of E2 conformation.

Structure and adsorption behavior of high hydrostatic pressure-treated β-lactoglobulin

HypothesisHigh hydrostatic pressure treatment causes structural changes in interfacial-active β-lactoglobulin (β-lg). We hypothesized that the pressure-induced structural changes affect the intra- and intermolecular interactions which determine the interfacial activity of β-lg. The conducted experimental and numerical investigations could contribute to the mechanistic understanding of the adsorption behavior of proteins in food-related emulsions. ExperimentsWe treated β-lg in water at pH 7 with high hydrostatic pressures up to 600 MPa for 10 min at 20 °C. The secondary structure was characterized with Fourier-transform infrared spectroscopy (FTIR) and circular dichroism (CD), the surface hydrophobicity and charge with fluorescence-spectroscopy and ζ-potential, and the quaternary structure with membrane-osmometry, analytical ultracentrifugation (AUC) and mass spectrometry (MS). Experimental analyses were supported through molecular dynamic (MD) simulations. The adsorption behavior was investigated with pendant drop analysis. FindingsMD simulation revealed a pressure-induced molten globule state of β-lg, confirmed by an unfolding of β-sheets with FTIR, a stabilization of α-helices with CD and loss in tertiary structure induced by an increase in surface hydrophobicity. Membrane-osmometry, AUC and MS indicated the formation of non-covalently linked dimers that migrated slower through the water phase, adsorbed more quickly due to hydrophobic interactions with the oil, and lowered the interfacial tension more strongly than reference β-lg.

Role of disulfide linkages in structure and activity of Kunitz trypsin inhibitor from Trigonella foenum-graecum (fenugreek) seeds and its porphyrin binding studies

Kunitz trypsin inhibitor (TfgKTI) isolated from fenugreek (Trigonella foenum-graecum) seeds inhibited the enzyme trypsin. The inhibitor is resistant to wide range of temperatures (37–90 °C) and pHs (3–10) but reduction of the inhibitor with dithiothreitol abolished trypsin inhibitory activity. Reduction of the disulfide linkages with dithiothreitol affected the tertiary structure significantly, whereas the secondary structure was not affected considerably. The kinetic plots of the reduction as followed by protein activity and loss in tertiary structure could be comparable. Chemical denaturation of TfgKTI with guanidine hydrochloride and fluorescence quenching studies by two neutral quenchers (acrylamide and succinimide), an anionic quencher Iodide ion (I−) and a cationic quencher Cesium ion (Cs+) suggested higher unfolding of protein under reduced conditions compared with native protein. Chemically denatured TfgKTI showed refolding, while it is irreversible with reduced protein. Native TfgKTI showed high binding affinity with porphyrin, but lost its binding capacity under reduced condition. Thus, disulfide linkages play a predominant role in maintaining the three-dimensional structure of the TfgKTI. The loss of inhibitory activity is correlated with loss in tertiary structure.

Effect of atmospheric cold plasma on structure, interfacial and emulsifying properties of Grass pea (Lathyrus sativus L.) protein isolate

Structure and emulsifying properties of Grass pea (Lathyrus sativus) protein isolate (GPPI) were investigated as function of cold plasma treatment at two voltages (9.4 and 18.6 kVpp) and treatment times (30s and 60s). Optical emission spectroscopy was used to characterize the nature of air DBD-cold plasma. The content of carbonyl group, Di-tyrosine cross-link and free sulfhydryl, secondary and tertiary structures, SDS-PAGE, surface charge, surface hydrophobicity and solubility of GPPI were characterized. Results from the optical emission showed that the density of charged and reactive species were greatly dependent on the voltage applied and increased at the higher voltage. With increasing the treatment time and voltage, the content of carbonyl groups, disulfide bonds, Di-tyrosine cross-link and also surface charge in GPPI increased, which in turn led to a decrease in the absorption rate of protein into the water-oil interface. However, higher efficiency of etching and oxidation rate at 18.6 kVpp after 60s of treatment, increased the dissociation of globulins, and thus the absorption rate of protein into interface increased. Plasma treatment resulted in the formation of more orderly secondary structures such as α and β structures. The loss of tertiary structure in the treated GPPIs at 9.4 kVpp increased the ability of GPPI in lowering the interfacial tension at oil-water interface. Whereas, more compact tertiary structure, higher ordered secondary structure and dissociation of globulins in the treated GPPI at 18.6 kVpp for 60s led to the formation of an interfacial layer with higher interfacial tension value. Compared to native GPPI, oil-droplets size of stabilized-emulsions with treated GPPI at 9.4 kVpp were bigger, indicating that these proteins were not able to form a strong interfacial film around the oil-droplets and prevent them from flocculation within the homogenizer. Whereas, in the emulsion stabilized with treated GPPIs at 18.6 kVpp for 60, the superior protein absorption and formation of dens and elastic interfacial film around oil droplets led to the formation of uniform small droplets with good stability against creaming. Overall, our results indicated that cold plasma treatment had positive effect on the interfacial and emulsifying properties of GPPI in terms of thermodynamic stability of protein on interface, globulin dissociation, and increase in oil-droplet surface electrical charge.

Low molecular weight α-galactosidase from black gram (Vigna mungo): Purification and insights towards biochemical and biophysical properties

A hitherto unknown low molecular weight form of α-galactosidase (VM-αGal-P) from germinating black gram (Vigna mungo) seeds was purified (324 U/mg specific activity, 1157-fold purification, ~45 kDa) using ion-exchange (DEAE-cellulose, CM-sepharose), gel filtration (Sephadex G-75) and affinity (Con-A Sepharose 4B) chromatography but with poor yield (0.75%). Partially purified enzyme (VM-αGal) (146.3 U/mg specific activity, 522.5-fold purification) was used for further studies. VM-αGal showed optimal activity at pH 5 and 55 °C. Hg2+ and SDS completely inhibited VM-αGal activity. The Km, Vmax and catalytic efficiency (kcat/Km) of VM-αGal for pNPG and raffinose was 0.99, 17.23 mM, 1.66, 0.146 μmol ml−1 min−1, and 0.413, 0.0026 s−1 mM−1, respectively. VM-αGal was competitively inhibited by galactose (Ki 7.70 mM). Thermodynamic parameters [activation enthalpy (ΔH), activation entropy (ΔS) and free energy (ΔG)] of VM-αGal at 45-51 °C showed that VM-αGal was in a less energetic state and had susceptibility towards denaturation. Temperature-induced structural unfolding studies of VM-αGal probed by fluorescence, and far-UV CD spectroscopy revealed significant loss in tertiary structure and a steep decline in β-sheet content at 45–65 °C, and above 55 °C, respectively. VM-αGal improved the nutritional quality of soymilk by hydrolyzing raffinose family oligosaccharides (26.5% and 18.45% decrease in stachyose and raffinose, respectively).

Sulfate dodecyl sodium-induced stability of a model intrinsically disordered protein, bovine casein

With well-known nutritional properties, casein contributes to about 80% of protein content in milk and has been classified as highly intrinsically disordered protein (IDP). In this paper, the sulfate dodecyl sodium (SDS)-induced conformational changes of bovine casein were studied by multi-techniques. Isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC) were used to obtain the stoichiometry of conformational changes and the thermal stability of the formed complexes. Spectral results indicated that casein presented a higher helical content but loss of tertiary structure above critical micelle concentration of SDS, namely, the so-called molten globule like state. The thermal self-association of casein could be prevented by SDS according to far-UV CD even at 70 °C. The 1H NMR spectrum of casein showed that the resonance around 1.0 ppm, the region of α-hydrogen, shifted to the higher field, and the aromatic region around 5.5–8.0 ppm shifted to the lower field, while the NOESY spectra of casein exhibited few chemical shifts with binding of SDS. Combining the results of dynamic light scattering (DLS), scanning electron microscope (SEM) and small angle x-ray scattering (SAXS), one obtains that casein micelles presented an elliptical shape of ∼800 nm in diameter and upon binding with SDS, the casein micelles disassociated into more compact globular particles of 10 nm in diameter with a core-shell structure composed by SDS molecules and casein proteins. The present work, not only provides molecular insights into the mechanism of SDS-induced stability of a model IDP, casein, but also helps understand the role of surfactants on the structure–function relationship of bovine casein in the food industry.

Cadherin Order and Dynamics in Calcium-Dependent and Independent Desmosomes

Desmosomes are complex cell-cell junctions composed of adhesive transmembrane cadherin proteins and intracellular plaque proteins. Integration of the desmosomal complex with the keratin intermediate filament cytoskeleton confers strength and mechanical resistance to epithelial tissue. Desmosomes can adopt different functional states in vivo that are critical for regulating adhesion during processes like wound healing and development. These states are characterized by desmosome sensitivity to calcium; upon reduction of extracellular calcium, cells with calcium-dependent desmosomes lose adhesion while cells with hyper-adhesive (calcium-independent) desmosomes maintain strong adhesion. Though defined by resistance to reduced calcium, the mechanism that confers hyper-adhesion is not known. Interestingly, the cadherin tertiary structure is dependent upon calcium binding for its rigidity. Furthermore, electron tomography showed desmosomal cadherins form an ordered array at the adhesive interface, and this order is hypothesized to be critical for adhesion. To address the mechanism driving hyper-adhesion, we investigated the order and dynamics of the desmosomal cadherin desmoglein 3 (Dsg3) in calcium dependent and hyper-adhesive states. Using fluorescence polarization microscopy, we found that Dsg3 was ordered in calcium-dependent desmosomes. When calcium was reduced, order was lost (τ= 5 min) faster than adhesive function was compromised (τ= 15 min). When we induced hyper-adhesion by inhibiting PKCα we similarly found Dsg3 was ordered. Surprisingly, when calcium was reduced in hyper-adhesive cells, order was lost with similar kinetics to the calcium-dependent cells, despite maintained adhesion. Measuring the order and dynamics of Dsg3 has revealed novel differences in ultrastructure between functional states in living cells. Our results demonstrate that desmosome adhesion is not directly dependent on cadherin order and cadherin trans-binding can persist after loss of tertiary structure. Because cadherins lose order regardless of adhesive state, these results suggest a central role for the desmosomal plaque in conferring hyper-adhesion.

Stabilizing vitamin D3 using the molten globule state of α-lactalbumin

α-Lactalbumin (α-LA) is the second most abundant bovine whey protein. It has been intensively studied because of its readiness to populate the molten globular (MG) state, a partially folded state with native levels of secondary structure but loss of tertiary structure. The MG state of α-LA exposes a significant number of hydrophobic patches that could be used to bind and stabilize small hydrophobic molecules such as vitamin D3 (vitD). Accordingly, we tested the ability of α-LA to stabilize vitD in a pH interval from 7.4 to 2; over this pH interval, α-LA transitions from the folded state to the MG state. The MG state stabilized vitD better than the folded state and was superior to the major bovine whey protein β-lactoglobulin (β-LG), which is known to stabilize vitD. At pH 7.4, β-LG and α-LA stabilized vitD to the same extent. Tryptophan fluorescence quenching measurements indicated that α-LA has one binding site at pH 7.4 but acquires an additional binding site when the pH is lowered to pH 2 to 4. Stability measurements of the vitD in the α-LA-vitD complex at different temperatures suggest that UHT processing would lead to little loss of vitD. This study demonstrates the potential of α-LA as a component in vitD fortification, particularly for low pH applications.

In vitro evaluation of the non-covalent interactions of hemoglobin with distinctively modified gemini surfactants: Effect of structural variation

Binding of surfactants to biomacromolecules is an active field of current interest at the interface of chemical biology and medicinal chemistry, owing to their diverse applications in industries, biomedical and cosmetic domains. In relevance to this, we have investigated the interactions of two distinctly modified green gemini surfactants (C12-E2O-C12 and C14-E2O-C14) with the heme protein hemoglobin (Hb) utilizing different state-of-the-art techniques. These surfactants are employed to explicate the effect of structural variation on the conformation of Hb. Complexation between Hb and C12-E2O-C12/C14-E2O-C14 were found to follow the same 1:1 stoichiometric pattern but with different binding constants (evaluated through fluorescence and electrochemical measurements). The UV–vis spectra showed the influence of C12-E2O-C12/C14-E2O-C14 on Hb, displaying a strong concentration-dependent fashion. Furthermore, pyrene, synchronous and 3-D fluorescence spectra of Hb depicted the possible alterations induced in its aromatic microenvironment upon C12-E2O-C12/C14-E2O-C14 complexation. Far-UV CD results revealed nominal changes in the secondary structure of the Hb while a considerable loss in tertiary structure was observed in the near-UV range, elucidating the formation of molten globule state. The best energy-docked structure in the molecular docking analysis revealed that C12-E2O-C12/C14-E2O-C14 preferred to bind in the hydrophobic cavity of Hb. Thus, this work provides a comprehensive inspection of Hb-C12-E2O-C12/C14-E2O-C14 interaction at the molecular level that can be extended potentially to other congeners, which is essential in determining their future use as excipients in pharmaceutical formulations and other related applications.

Insight into the co-solvent induced conformational changes and aggregation of bovine β-lactoglobulin

Many proteins form ordered irreversible aggregates called amyloid fibrils which are responsible for several neurodegenerative diseases. β-lactoglobulin (β-lg), an important globular milk protein, self-assembles to form amyloid-like fibrils on heating at low pH. The present study investigated the effects of two commonly used organic solvents acetonitrile (MeCN) and antimicrobial preservative benzyl alcohol (BA) on the conformation and self-assembly of β-lg at ambient condition. Both MeCN and BA induced a concentration-dependent conformational change showing exposure of hydrophobic patches, loss of tertiary structure and higher α-helical structure at moderate concentrations. In the presence of 50–80% (v/v) MeCN and 1.5–3% (v/v) BA further structural transitions from α-helical to non-native β-sheet structure were observed with a molten globule-like intermediate at 70% MeCN. These non-native β-sheet structures have high tendency to form aggregates. The formation of β-lg self-assembly was confirmed by Thioflavin T studies, Congo red assay, Rayleigh scattering and dynamic light scattering analysis. Transmission electron microscopy studies showed amyloid fibril formation in both MeCN and BA. Our results showed that BA enhances the unfolding and self-assembly of β-lg at much lower concentration than MeCN. Thus solvent composition forces the protein to achieve the non-native structures which are responsible for protein aggregation.

Stability of Curcuma longa rhizome lectin: Role of N-linked glycosylation.

Curcuma longa rhizome lectin, a mannose-binding protein of non-seed portions of turmeric, is known to have antifungal, antibacterial and α-glucosidase inhibitory activities. We studied the role of complex-type glycans attached to asparagine (Asn) 66 and Asn 110 to elucidate the role of carbohydrates in lectin activity and stability. Apart from the native lectin, the characteristics of a deglycosylated Escherichia coli expressed lectin, high-mannose oligosaccharides at both asparagines and its glycosylation mutants N66Q and N110Q expressed in Pichia pastoris, were compared to understand the relationship between glycosylation and activity. Far UV circular dichroism (CD) spectra, fluorescence emission maximum, hemagglutination assay show no change in secondary or tertiary structures or sugar-binding properties between wild-type and aforementioned recombinant lectins under physiological pH. But reduced agglutination activity and loss of tertiary structure are observed in the acidic pH range for the deglycosylated and the N110Q protein. In thermal and guanidine hydrochloride (GdnCl)-induced unfolding, the wild-type and high-mannose lectins possess higher stability compared with the deglycosylated recombinant lectin and both mutants, as measured by a higher Tm of denaturation or a greater free energy change, respectively. Reversibility experiments after thermal denaturation reveal that deglycosylated proteins tend to aggregate during thermal inactivation but the wild type shows a much greater recovery to the native state upon refolding. These results suggest that N-glycosylation in turmeric lectin is important for the maintenance of its proper folding upon changes in pH, and that the oligosaccharides help in maintaining the active conformation and prevent aggregation in unfolded or partially folded molecules.

The stability of the TIM-barrel domain of a psychrophilic chitinase

Chitinase 60 from the psychrophilic bacterium Moritella marina (MmChi60) is a four-domain protein whose structure revealed flexible hinge regions between the domains, yielding conformations in solution that range from fully extended to compact. The catalytic domain is a shallow-grooved TIM-barrel. Heat-induced denaturation experiments of the wild-type and mutants resulting from the deletions of the two-Ig-like domains and the chitin binding domain reveal calorimetric profiles that are consistent with non-collaborative thermal unfolding of the individual domains, a property that must be associated to the “hinge-regions”. The calorimetric measurements of the (β/α)8 catalytic domain reveal that the thermal unfolding is a slow-relaxation transition exhibiting a stable, partially structured intermediate state. Circular dichroism provides evidence that the intermediate exhibits features of a molten globule i.e., loss of tertiary structure while maintaining the secondary structural elements of the native. GdnHCl-induced denaturation studies of the TIM-barrel demonstrate an extraordinarily high resistance to the denaturant. Slow-relaxation kinetics characterize the unfolding with equilibration times exceeding six days, a property that is for the first time observed for a psychrophilic TIM barrel. On the other hand, the thermodynamic stability is ΔG=6.75±1.3kcal/mol, considerably lower than for structural-insertions-containing barrels. The mutant E153Q used for the crystallographic studies of MmChi60 complexes with NAG ligands has a much lower stability than the wild-type.

Structural elucidation and molecular characterization of Marinobacter sp. α-amylase

ABSTRACTHalophiles have been perceived as potential source of novel enzymes in recent years. The interest emanates from their ability to catalyze efficiently under high salt and organic solvents. Marinobacter sp. EMB8 α-amylase was found to be active and stable in salt and organic solvents. A study was carried out using circular dichroism (CD), fluorescence spectroscopy, and bioinformatics analysis of similar protein sequence to ascertain molecular basis of salt and solvent adaptability of α-amylase. Structural changes recorded in the presence of varying amounts of NaCl exhibited an increase in negative ellipticity as a function of salt, confirming that salt stabilizes the protein and increases the secondary structure, making it catalytically functional. The data of intrinsic and extrinsic fluorescence (using 1-anilinonaphthalene 8-sulfonate [ANS] as probe) further confirmed the role of salt. The α-amylase was active in the presence of nonpolar solvents, namely, hexane and decane, but inactivated by ethanol. The decrease in the activity was correlated with the loss of tertiary structure in the presence of ethanol. Guanidine hydrochloride and pH denaturation indicated the molten globule state at pH 4.0. Partial N-terminal amino acid sequence of the purified α-amylase revealed the relatedness to Pseudoalteromonas sp. α-amylase. “FVHLFEW” was found as the N-terminal signature sequence. Bioinformatics analysis was done using M. algicola α-amylase protein having the same N-terminal signature sequence. The three-dimensional structure of Marinobacter α-amylase was deduced using the I-TASSER server, which reflected the enrichment of acidic amino acids on the surface, imparting the stability in the presence of salt. Our study clearly indicate that salt is necessary for maintaining the secondary and tertiary structure of halophilic protein, which is a necessary prerequisite for catalysis.

Bovine lactoferrin binds oleic acid to form an anti-tumor complex similar to HAMLET

α-Lactalbumin (α-LA) can bind oleic acid (OA) to form HAMLET-like complexes, which exhibited highly selective anti-tumor activity in vitro and in vivo. Considering the structural similarity to α-LA, we conjectured that lactoferrin (LF) could also bind OA to obtain a complex with anti-tumor activity. In this study, LF–OA was prepared and its activity and structural changes were compared with α-LA–OA. The anti-tumor activity was evaluated by methylene blue assay, while the apoptosis mechanism was analyzed using flow cytometry and Western blot. Structural changes of LF–OA were measured by fluorescence spectroscopy and circular dichroism. The interactions of OA with LF and α-LA were evaluated by isothermal titration calorimetry (ITC). LF–OA was obtained by heat-treatment at pH8.0 with LD50 of 4.88, 4.95 and 4.62μM for HepG2, HT29, and MCF-7 cells, respectively, all of which were 10 times higher than those of α-LA–OA. Similar to HAMLET, LF–OA induced apoptosis in tumor cells through both death receptor- and mitochondrial-mediated pathways. Exposure of tryptophan residues and the hydrophobic regions as well as the loss of tertiary structure were observed in LF–OA. Besides these similarities, LF showed different secondary structure changes when compared with α-LA, with a decrease of α-helix and β-turn and an increase of β-sheet and random coil. ITC results showed that there was a higher binding number of OA to LF than to α-LA, while both of the proteins interacted with OA through van der Waals forces and hydrogen bonds. This study provides a theoretical basis for further exploration of protein–OA complexes.

Molten Globule of Hemoglobin Proceeds into Aggregates and Advanced Glycated End Products

Conformational alterations of bovine hemoglobin (Hb) upon sequential addition of glyoxal over a range of 0–90% v/v were investigated. At 20% v/v glyoxal, molten globule (MG) state of Hb was observed by altered tryptophan fluorescence, high ANS binding, existence of intact heme, native-like secondary structure as depicted by far-UV circular dichroism (CD) and ATR-FTIR spectra as well as loss in tertiary structure as confirmed by near-UV CD spectra. In addition, size exclusion chromatography analysis depicted that MG state at 20% v/v glyoxal corresponded to expanded pre-dissociated dimers. Aggregates of Hb were detected at 70% v/v glyoxal. These aggregates of Hb had altered tryptophan environment, low ANS binding, exposed heme, increased β-sheet secondary structure, loss in tertiary structure, enhanced thioflavin T (ThT) fluorescence and red shifted Congo Red (CR) absorbance. On incubating Hb with 30% v/v glyoxal for 0–20 days, advanced glycation end products (AGEs) were detected on day 20. These AGEs were characterised by enhanced tryptophan fluorescence at 450 nm, exposure of heme, increase in intermolecular β-sheets, enhanced ThT fluorescence and red shift in CR absorbance. Comet assay revealed aggregates and AGEs to be genotoxic in nature. Scanning electron microscopy confirmed the amorphous structure of aggregates and branched fibrils of AGEs. The transformation of α-helix to β-sheet usually alters the normal protein to amyloidogenic resulting in a variety of protein conformational disorders such as diabetes, prion and Huntington's.

Top‐down structural analysis of posttranslationally modified proteins by Fourier transform ion cyclotron resonance‐MS with hydrogen/deuterium exchange and electron capture dissociation

High-resolution structural characterization of posttranslationally modified proteins represents a challenge for traditional structural biology methods such as crystallography and NMR. In this study, we have used top-down hydrogen/deuterium exchange MS (HDX-MS) with precursor ion selection and electron capture dissociation to determine the impact of oxidative modification on calmodulin (CaM) at an average resolution of 2.5 residues, with complete sequence coverage. The amide deuteration status of native CaM determined by this method correlates well with previously reported crystallographic and NMR data. In contrast, methionine oxidation caused almost complete deuteration of all residues in the protein in 10 s. The oxidative-modification-induced secondary and tertiary structure loss can be largely recovered upon calcium ligation, which also resulted in a substantial increase of amide protection in three of the four calcium-binding loops in oxidatively modified CaM (CaMox ). However, the structure of α-helix VI is not restored by cofactor binding. These results are discussed in terms of different target binding and activation capabilities displayed by CaM and CaMox . The isoform-specific top-down HDX structural analysis strategy demonstrated in this study should be readily applicable to other oxidatively modified proteins and other types of PTMs, and may help decipher the structure and function of specific protein isoforms.

Peroxynitrite-modified H3 Histone is Highly Immunogenic and Binds Circulating SLE Autoantibodies Better than Native DNA

Peroxynitrite is a bifaceted reactive species. It can oxidize and nitrate proteins/nucleic acids/lipids and is involved in inflammation, apoptosis, cytotoxicity and autoimmune disorders of unknown etiology, including SLE. In this study, H3 histone exposed to peroxynitrite caused loss of tertiary structure, nitration of tyrosine residues and dityrosine formation. Experimentally produced antibodies against peroxynitrite-modified H3 histone showed specificity for the immunogen. However, cross-reactions with other nitrated proteins were also observed. We further investigated the binding of SLE autoantibodies with native DNA, native H3 histone and peroxynitrite-modified H3 histone to explore the possible role of modified-H3 histone in the initiation and/or progression of SLE in a sub-group of patients. The results showed preferential binding of SLE anti-nDNA autoantibodies to peroxynitrite-modified H3 histone. The visual detection of immune complex formation with native and peroxynitrite-modified H3 histone reiterated preferential binding of SLE autoantibodies with modified H3 histone. It may be concluded that anti-histone autoantibodies seen in a subgroup of SLE patients might be due to the immunogenicity of peroxynitrite-modified H3 histone.

Non-native States of Bovine Beta-Lactoglobulin Induced by Acetonitrile: pH-Dependent Unfolding of the Two Genetic Variants A and B

Acetonitrile (ACN)-induced unfolding of the beta-lactoglobulin variants A and B was investigated at pH 2.0, 7.0 and 9.0. ACN caused α-helix induction at low concentrations but lead to major conformational alterations when the concentration was raised. ACN also induced a concentration-dependent increase in the surface hydrophobicity of both the variants. Induction of α-helical structure and exposure of hydrophobic patches were, however, somewhat more pronounced in case of variant B, whereas the loss of tertiary structure was more marked for variant A. Both protein aggregation and helix induction necessitated higher ACN concentrations at pH 2.0 than at 7.0 and 9.0, suggesting the greater stability of the variants at acidic pH.

Equilibrium studies of cellulase aggregates in presence of ascorbic and boric acid

The aggregate formation of cellulase was detected at 300 and 10mM ascorbic and boric acid respectively. These aggregates showed reduced enzyme activity, loss in near-UV signal, decrease tryptophan and ANS fluorescence. They possess increase in non-native β-sheet structure as evident from far-UV CD and FTIR spectra, large hydrodynamic radii, increase thioflavin T fluorescence and shift in Congo red. Cellulase at 90mM ascorbic acid exists as molten globule with retention of secondary structure, altered tryptophan environment, high ANS binding and loss in tertiary structure. Ascorbic acid acts as an antioxidant up to 90mM and beyond this as a pro-oxidant.

Controlling the morphological evolution of ZnO NPs from single precursor source and its application for β-lactoglobulin adsorption

Control over the synthesis of morphologically different ZnO nanoparticles (NPs), using same precursor aromatic organic acid as ligand for Zn2+ co-ordination, has been reported in this study. Pyridine-2,3-dicarboxylic acid (PDC) was used as a ligand to form Zn(II)–PDC precursor complexes of different architectures, which on further calcination at 600 °C produce ZnO NPs of different morphology. The monomer Zn(II)–PDC complex leads to disk-like ZnO NPs of ~30 nm diameter, whereas the co-ordination polymer of Zn(II)–PDC when heated predominantly produce ZnO nanorod of length ~100 nm. We have also investigated the bio-interaction of these ZnO NPs with β-lactoglobulin (β-LG) adsorption study. Significantly, differential binding behavior of β-LG on these two morphologically different nanostructures has been obtained. The protein interaction process was found to be pH-dependent on disk-like ZnO NPs though thermodynamically favorable under all studied condition. The thorough analysis of the adsorption process has been performed by varying the factors such as protein and NP concentration, temperature, time, and pH. In-depth structural analysis of the protein interacting with ZnO NPs has been done using spectroscopic techniques. Fluorescence emission and anisotropy analysis reveals a partial loss in tertiary structure of β-LG upon binding on surface. However, the circular dichroism spectra show no significant change in the secondary component upon adsorption.