Impact of metal nanoparticles on the structure and function of metabolic enzymes.

The widespread use of nanoparticles raises many serious concerns about the safety and environmental impact of nanoparticles. Therefore, risk assessments of specific nanoparticles in occupational and environmental exposure are essential before their large-scale production and applications, especially in medicine and for usage in household items. In this study, the effects of five different metal nanoparticles on the structure, stability, and function of four metabolic enzymes were evaluated using various biophysical techniques. Our results show that Cu nanoparticles exhibited the most significant adverse effects on the structures, stability, and activities of all the metabolic enzymes. Zn nanoparticles caused moderate adverse effects on these enzymes. The rest of the metal (Al, Fe, and Ni) nanoparticles had a relatively lower impact on the metabolic enzymes. Our data indicated that Cu nanoparticles promote metal-catalyzed disulfide bond formation in these proteins. In summary, some metal nanoparticles can cause adverse effects on the structure, function, and stability of metabolic enzymes. In addition, metal nanoparticles may affect protein homeostasis in the cytosol or extracellular fluids.

Effect of Antioxidants on Heavy Metals Induced Conformational Alteration of Cytochrome C and Myoglobin.

BACKGROUND: The exposure to heavy metals due to unrestrained industrialization, pollution and non-degradability imposes a significant risk to human health. Proteins are prime targets of heavy metal stress, however, the underlying mechanisms and its impact on heme proteins is still not entirely clear. OBJECTIVE: To analyze the deleterious effect of heavy metals such as cadmium, chromium and mercury on conformation of two proteins namely, cytochrome c and myoglobin. The protective effect of glycine and ascorbic acid (animal origin), gallic acid and sesamol (plant origin) on heavy metal exposure was studied. METHODS: Far- and near-UV Circular Dichroism (CD) measurements monitored the changes in secondary and tertiary structure. Absorption Soret spectroscopy study revealed changes in heme-protein interaction. Peroxidase activity has been assayed to measure the absorption of tetraguaiacol. The interaction of heme proteins with different heavy metals was done using docking study. RESULTS: Far- and near-UV CD measurements reveal that heavy metals disrupt the secondary and tertiary structure of heme proteins. Antioxidants counteract the deleterious effect of heavy metals. Absorption spectroscopy revealed changes in the Soret region of these heme proteins. Changes in peroxidase activity was observed on addition of heavy metals and antioxidants. Molecular docking validated interaction of the heavy metals with proteins with a significant binding affinity (-2.3 kcal/- mol). CONCLUSION: Heavy metals interfered and disrupted both the heme proteins and mercury showed the maximum deleterious effect, further, chromium showed detrimental effect at very small concentration. The antioxidants from animal origin exhibited better protective response than those from plant source.

Protection of ζ-crystallin by α-crystallin under thermal stress.

Cataract is one of the major causes of blindness worldwide. Several factors including post-translational modification, thermal and solar radiations promote cataractogenesis. The camel lens proteins survive very harsh desert conditions and resist cataractogenesis. The folding and aggregation mechanism of camel lens proteins are poorly characterized. The camel lens contains three ubiquitous crystallins (α-, ß-, and γ-crystallin) and a novel protein (ζ-crystallin) in large amounts. In this study, a sequence similarity search of camel α-crystallin with that of other organisms showed that the camel αB-crystallin consists of an extended N-terminal domain. Our results indicate that camel α-crystallin efficiently prevented aggregation of ζ-crystallin, with or without an obligate cofactor up to 89 °C. It performed a quick and efficient holdase function irrespective of the unfolding stage or aggregation. Camel α-crystallin exhibits approximately 20% chaperone activity between 30 and 40 °C and is completely activated above 40 °C. Camel α-crystallin underwent a single reversible thermal transition without loss of ß-sheet secondary structure. Intrinsic tryptophan fluorescence and ANS binding experiments revealed two transitions which corresponded to activation of its chaperone function. In contrast to earlier studies, camel α-crystallin completely protected lens proteins during thermal stress.

Circulation of dengue virus serotypes in hyperendemic region of New Delhi, India during 2011-2017.

BACKGROUND: Dengue fever has become a hampering menace in New Delhi India, since the disease has become hyperendemic, due to circulation of multiple serotypes of dengue virus (DENV). This hyperendemicity poses a greater risk of secondary infections in human health system. This is a major issue which leads to apprehension amongst the researchers and health organizations and thus requires regular epidemiological surveillance. METHODS: We analyzed the prevalence and serotypic distribution of dengue fever cases reported from the Southern part of New Delhi during continued surveillance from 2011 to 2017. The blood samples for the investigation were obtained from the patients suspected with dengue fever attending the OPD at a local Health Centre. The data for 2011-2016 was already published from our laboratory. The samples collected during 2017 were serotyped and characterized in the present study. RESULTS: A total of 565 samples (59%) were positive for DENV of 958 samples tested by RT-PCR during 7 years (2011-2017). Our study has shown that most infections were caused by DENV-2 during 2011-2015. The data has shown occurrence of all four serotypes of DENV during 2015 and predominance of DENV-3 in 2016 and 2017. Further, predominant combination of DENV-1 and DENV-2 was found in most of the co-infections. To the best of our knowledge this is the first study showing the epidemiological trend of dengue fever in reference to the circulating DENV serotypes and co-infections from a hyperendemic region of New Delhi during 2011-2017. CONCLUSIONS: This hyperendemic pattern of DENV and instantaneous shift in circulation of its serotypes is likely pose a greater risk of secondary infections. Inclusion of comprehensive community and hospital surveillance of dengue fever will assist in formulation and implementation of effective control measures.

Structural stability and solubility of glycated camel lens ζ-crystallin.

The camel has several biochemical, physiological, and anatomical features to withstand the harsh desert climate. Camel eye lens contains a novel protein (ζ-crystallin) in bulk quantity. Previous reports suggest that non-enzymatic glycation of eye lens proteins plays an important role in the etiology of cataract. In this study, we have characterized the role of glucose, fructose, and methylglyoxal (MGO) in the glycation of camel lens ζ-crystallin. From the results obtained, it was found that MGO reacted rapidly, fructose reacted moderately, and glucose was the least reactive even after prolonged incubation (>100 days). Glycation with MGO and fructose led to changes in the structure of ζ-crystallin, while glucose had no remarkable effect. The surface hydrophobicity did not change and no aggregates or amyloid fibrils were observed in the glycated ζ-crystallin. Moreover, the secondary structure of glycated ζ-crystallin remained similar after glycation. Our results suggested that due to natural adaptation, the camel lens protein ζ-crystallin retained its structure and solubility even after glycation to perform the single known function of the lens proteins: to focus unscattered light on the retina.

An efficient methodology for the purification of date palm peroxidase: Stability comparison with horseradish peroxidase (HRP).

In the present study, Peroxidase from date palm (Phoenix dactylifera) leaves was purified to homogeneity by three-step procedure including aqueous two-phase system, hydrophobic and Ion-exchange chromatography. The enzyme migrated as single band on SDS-PAGE giving molecular weight of 68 ±â€¯3 kDa. The purification factor for purified date palm peroxidase was 68 with high 41% yield. Enzymatic assays together with far-UV circular dichroism (CD), intrinsic and extrinsic fluorescence studies were carried out to monitor the structural stability of date palm and horseradish peroxidase (HRP) against various pH and temperatures. Activity measurements illustrated different pH stability for date palm and HRP. Both peroxidases are more susceptible to extreme acidic conditions as suggested by 4 & 15 nm red shift in date palm and HRP, respectively. Secondary structure analysis using far UV-CD exhibited predominance of α-helical (43.8%) structure. Also, pH induces loss in the secondary structure of date palm peroxidase. Thermal stability analysis revealed date palm peroxidase is more stable in comparison to HRP. In summary, date palm peroxidases could be promising enzymes for various applications where extreme pH and temperature is required.

Effect of cetyltrimethylammonium bromide (CTAB) on the conformation of a hen egg white lysozyme: A spectroscopic and molecular docking study.

The interactions between cetyltrimethylammonium bromide (CTAB) and hen egg white lysozymes (HEWL) was carried out to investigate protein-surfactant interaction mechanisms while both exist in the overall same charged state. The interactions between CTAB and the HEWL were examined with circular dichroism (CD), dynamic light scattering (DLS), fluorescence spectroscopy, and computational docking at a pH9.0 at room temperature. The far-UV CD and fluorescence results revealed that CTAB at concentrations from 0.15 to 10.0mM influenced the secondary as well as the tertiary structure of HEWL. The secondary structure of the HEWL was retained, while the tertiary structure of the HEWL was disrupted in the CTAB-treated samples at pH9.0. The hydrodynamic radii of the HEWL were also expanded in the presence of CTAB. Molecular docking studies showed that CTAB formed one electrostatic and four hydrophobic interactions, as well as one carbon hydrogen bond with HEWL. The data obtained from spectroscopic and computational studies demonstrated that the positively charged head and 18­carbon alkyl chain of the CTAB interacted through weak electrostatic and strong hydrophobic interactions.

Food additive dye (quinoline yellow) promotes unfolding and aggregation of myoglobin: A spectroscopic and molecular docking analysis.

Protein aggregation leads to vast conformational changes and plays a key role in the pathogenesis of various neurodegenerative diseases including Alzheimer's and Parkinson's. In the current piece of work, we have explored the interaction of quinoline yellow (QY) with myoglobin (Mb) at two different pH (3.5 and 7.4). Various spectroscopic techniques such as turbidity, Rayleigh light scattering (RLS), UV-Vis absorbance, fluorescence resonance energy transfer (FRET), far UV-CD along with transmission electron microscopy (TEM) and molecular docking have been utilized to characterize dye-induced aggregation in Mb. Binding results showed that interaction between QY and myoglobin is spontaneous and static in nature with high KSV value of 2.14 × 104 M-1. On the other hand, thermodynamics studies (∆H & ∆S) revealed that complex formation was driven by hydrogen and Van der Walls forces. Molecular docking analysis showed strong binding affinity (Kd = 4.95 × 104 M-1) between QY and Mb at Pro100, Ile101, Lys102, Glu105, Glu136, Arg139, Lys140, and Ala143 residues. The intrinsic fluorescence and circular dichroism studies indicated that QY induced conformational changes in Mb at pH 3.5. Turbidity and RLS studies showed aggregation of Mb in the presence of QY (0.2-5 mM). Moreover, kinetics data revealed nucleation independent aggregation of myoglobin in the presence of QY. TEM analysis further established amorphous nature of Mb aggregate induced by QY. At pH (7.4), QY was unable to induce aggregation in myoglobin; it might be due to repulsive nature of negatively charged dye and myoglobin or partially altered states of protein could be pre-requisite for binding and aggregation.

Different conformational states of hen egg white lysozyme formed by exposure to the surfactant of sodium dodecyl benzenesulfonate.

The aim of this study was to investigate the effects of sodium dodecyl benzenesulfonate (SDBS) on hen egg white lysozyme (HEWL) fibrillogenesis at pH 7.4. HEWL fibrillogenesis in the presence of SDBS was characterized using several spectroscopic techniques (turbidity, light scattering, intrinsic fluorescence, ThT binding assay, ThT kinetics, far-UV CD, and transmission electron mmicroscopy). The turbidity and light scattering data revealed that SDBS induces aggregation in HEWL in dose-dependent manner. HEWL aggregation was seen at low SDBS concentrations (0.03 to 0.5 mM) but it was not observed at concentrations of SDBS at >0.6 mM. The ThT and TEM data clearly showed that the aggregates formed in the presence of SDBS had an amyloid-like morphology. From the CD analysis it was clear that low SDBS concentrations decreases the α-helical content while the ß-sheet content increased. As the SDBS concentration further increased, the α-helical content increased again. The ThT kinetics analysis revealed that the HEWL monomer directly converted into the amyloid fibril without lag phase. All the spectroscopic and microscopic results support the finding that low concentrations of SDBS stimulate fibrillogenesis in HEWL, and that no fibrillogenesis occurs at higher SDBS concentrations.

Allura red rapidly induces amyloid-like fibril formation in hen egg white lysozyme at physiological pH.

Allura red (AR) is an artificial azo dye mostly used in food industries and has potential health risks. We examined the role of AR in amyloidogenesis using hen egg white lysozyme (HEWL) at pH 7.0. The amyloidogenic induction properties of AR in HEWL were identified by circular dichroism (CD), turbidity, intrinsic fluorescence, light scattering, transmission electron microscopy (TEM), and molecular dynamic simulation studies. Turbidity and light scattering measurements showed that HEWL becomes aggregated in the presence of 0.03-15.0 mM of AR at pH 7.0 but not at very low AR concentrations (0.01-0.28 mM). However, AR-induced aggregation is a kinetically rapid process, with no observable lag phase and saturation within 6 s. The kinetics results suggested that the HEWL aggregation induced by AR is very rapid. The CD results demonstrated that the total ß-sheet content of HEWL was increased in the AR treated samples. The TEM results are established that AR-induced aggregates had amyloid-like structures. Molecular dynamics simulations analysis showed that the bound AR-HEWL structures were highly favored compared to unbound structures. The mechanism of AR-induced amyloid fibril formation may involve electrostatic, hydrogen bonding, and hydrophobic interactions.

Rutin attenuates negatively charged surfactant (SDS)-induced lysozyme aggregation/amyloid formation and its cytotoxicity.

Amyloid fibrils are highly ordered protein assemblies known to contribute to the pathology of a variety of genetic and aging-associated diseases. Here, we have investigated the aggregation propensity of lysozyme in the presence of a negatively charged surfactant (SDS) and evaluated the anti-aggregation activity of rutin. Multiple approaches such as turbidity measurements, dye binding assays, intrinsic fluorescence, circular dichroism (CD), transmission electron microscopy (TEM), MTT and comet assays have been used for this purpose. We inferred that SDS induces aggregation of lysozyme in 0.2-0.6 mM concentration range while at higher concentration range (0.8-1.0 mM), it leads to solubilization/stabilization of protein. Intrinsic/extrinsic fluorescence and CD analysis confirmed significant conformational changes in lysozyme at 0.2 mM SDS. Thioflavin T (ThT), congo red binding and TEM analysis further reaffirmed the formation of lysozyme fibrils. Moreover, MTT assay demonstrated cytotoxicity of these fibrils towards neuroblastoma cell lines (SH-SY5Y) and their attenuation by rutin. Comet assay supported the cytotoxicity mechanism via DNA damage. Molecular docking results also advocate a strong interaction between lysozyme and rutin. The current study indicates a mechanistic approach assuming structural constraints and specific aromatic interactions of rutin with HEWL aggregates.