Sadaf Fatima

pH-Induced Molten Globule State of Rhizopus niveus Lipase is More Resistant Against Thermal and Chemical Denaturation Than Its Native State

Here, we have characterized four pH-dependent states: alkaline state, “B” (pH 9.0), native state, “N” (pH 7.4), acid-induced state, “A” (pH 2.2) and molten globule state, “MG” (pH 1.8) of Rhizopus niveus lipase (RNL) by CD, tryptophanyl fluorescence, ANS binding, DLS, and enzyme activity assay. This “MG” state lacks catalytic activity and tertiary structure but it has native-like significant secondary structure. The “Rh” of all the four states of RNL obtained from DLS study suggests that the molecular compactness of the protein increases as the pH of solution decreases. Kinetic analysis of RNL shows that it has maximum catalytic efficiency at state “B” which is 15-fold higher than state “N.” The CD and tryptophanyl fluorescence studies of RNL on GuHCl and temperature-induced unfolding reveal that the “MG” state is more stable than the other states. The DSC endotherms of RNL obtained at pH 9.0, 7.4, and 2.2 were with two transitions, while at pH 1.8 it showed only a single transition.

Interactions of thioflavin T with serum albumins: Spectroscopic analyses

The interaction of thioflavin T (ThT) with serum albumins from four different mammalian species i.e. human, bovine, porcine and rabbit, has been investigated by circular dichroism (CD), fluorescence spectroscopy and ITC. The binding constant (K) for HSA was found to be 9.9 x 10(4)M(-1), 4.3 x 10(4)M(-1) for RSA, 1.07 x 10(4)M(-1) for PSA and 0.3 x 10(4)M(-1) for BSA and the number of binding sites (n) were 1.14, 1.06, 0.94 and 0.8, respectively, which is very significant. By using unfolding pathway of HSA in the presence of urea, domain II of HSA has been assigned to possess binding site of ThT. Its binding constant is comparable to many drugs that bind at domain II of HSA, like salicylate, warfarin, digitoxin, etc. Acting force between HSA and ThT is showing that both hydrophobic and electrostatic forces have contributed for the interaction. DeltaG(binding), DeltaH and DeltaS were calculated to be -28.46 kJ mol(-1), -3.50 kJ mol(-1) and 81.04 JK(-1)mol(-1), respectively. The data described here will help to increase our understanding about the interaction of ThT with native proteins. The results also indicate that care must be taken while using ThT as a probe for detecting amyloid fibrils.

More stable structure of wheat germ lipase at low pH than its native state.

Wheat germ lipase is a cereal lipase which is a monomeric protein. In the present study we sought to structurally characterize this protein along with equilibrium unfolding in solution. Conformational changes occurring in the protein with varying pH, were monitored by circular dichroism (CD) spectroscopy, fluorescence emission spectroscopy, binding of hydrophobic dye, 1-anilino 8-naphthalenesulfonic acid (ANS) and dynamic light scattering (DLS). Our study showed that acid denaturation of lipase lead to characterization of multiple monomeric intermediates. Native protein at pH 7.0 showed far-UV spectrum indicating mixed structure with both alpha and beta-type of characteristics. Activity of lipase was found to fall on either sides of pH 7.0-8.0. Acid-unfolded state was characterized at pH 4.0 with residual secondary structure, disrupted tertiary spectrum and red-shifted fluorescence spectrum with decreased intensity. Further decrease in pH lead to formation of secondary structure and acid-induced molten globule state was found to be stabilized at pH 1.4, with exposed tryptophan residues and hydrophobic patches. Notably, interesting finding of this study was characterization of acid-induced state at pH 0.8 with higher secondary structure content than native lipase, regain in tertiary spectrum and induction of compact conformation. Although enzymatically inactive, acid-induced state at pH 0.8 was found to be structurally more stable than native lipase, as shown by chemical and thermal denaturation profiles.

Measuring Glutathione Redox Potential of HIV-1-infected Macrophages

Background: Abnormal glutathione poise has been associated with HIV-1 infection; however, the underlying importance is unclear. Results: Measurement of glutathione-redox potential (EGSH) revealed higher capacity of latently infected cells to resist oxidative stress and apoptosis, whereas HIV-1 replication perturbed glutathione homeostasis. Conclusion: Dynamic changes in EGSH regulate HIV-1 persistence and reactivation. Significance: Glutathione-redox signaling plays a critical role in HIV-1 infection. Redox signaling plays a crucial role in the pathogenesis of human immunodeficiency virus type-1 (HIV-1). The majority of HIV redox research relies on measuring redox stress using invasive technologies, which are unreliable and do not provide information about the contributions of subcellular compartments. A major technological leap emerges from the development of genetically encoded redox-sensitive green fluorescent proteins (roGFPs), which provide sensitive and compartment-specific insights into redox homeostasis. Here, we exploited a roGFP-based specific bioprobe of glutathione redox potential (EGSH; Grx1-roGFP2) and measured subcellular changes in EGSH during various phases of HIV-1 infection using U1 monocytic cells (latently infected U937 cells with HIV-1). We show that although U937 and U1 cells demonstrate significantly reduced cytosolic and mitochondrial EGSH (approximately −310 mV), active viral replication induces substantial oxidative stress (EGSH more than −240 mV). Furthermore, exposure to a physiologically relevant oxidant, hydrogen peroxide (H2O2), induces significant deviations in subcellular EGSH between U937 and U1, which distinctly modulates susceptibility to apoptosis. Using Grx1-roGFP2, we demonstrate that a marginal increase of about ∼25 mV in EGSH is sufficient to switch HIV-1 from latency to reactivation, raising the possibility of purging HIV-1 by redox modulators without triggering detrimental changes in cellular physiology. Importantly, we show that bioactive lipids synthesized by clinical drug-resistant isolates of Mycobacterium tuberculosis reactivate HIV-1 through modulation of intracellular EGSH. Finally, the expression analysis of U1 and patient peripheral blood mononuclear cells demonstrated a major recalibration of cellular redox homeostatic pathways during persistence and active replication of HIV.

Petrographic and geochemical characteristics of Mesoproterozoic Kumbalgarh clastic rocks, NW Indian shield: implications for provenance, tectonic setting, and crustal evolution

The Kumbalgarh Group of the south Delhi fold belt are the main bedrock series exposed in the axial region of the Aravalli craton. Quartzites and greywackes, the chief clastic constituents of this group, are well exposed. Petrographic and bulk-rock analyses of these rocks permit determination of their provenance, tectonic setting of the basin, and the Archaean to Proterozoic crustal evolution. Greywackes comprise quartz, plagioclase, amphiboles, K-feldspar, and rock fragments. Based on mineralogy, we divided the quartzites into three categories: QTZ1 is chiefly composed of quartz with a silty matrix and a minor quantity of feldspars and QTZ2 contains significant mafic minerals as well as quartz and feldspars, whereas QTZ3 is more feldspathic than the other groups. All the lithounits have SiO2/Al2O3 ratios <∼10 suggesting textural immaturity consistent with their sedimentary petrography. Greywackes display the least fractionated rare earth elements (REEs) (La/Yb N : avg. 2.55) with positive Eu anomalies (avg. Eu/Eu* = 1.34). QTZ1 contains strongly fractionated REE patterns (avg. La/Yb N : 13.56, avg. Eu/Eu* = 0.60), QTZ2 shows moderate REE fractionation (avg. La/Yb N : 4.97, avg. Eu/Eu* = 0.61), and QTZ3 possesses the least fractionated V-shaped REE patterns (avg. La/Yb N : 1.97, avg. Eu/Eu* = 0.51). Weathering attributes including chemical index of alteration (CIA), plagioclase index of alteration (PIA), chemical index of weathering (CIW), and A–CN–K plots assign a low to moderate degree of weathering to the Kumbalgarh sediments under a subtropical climate. Based on our synthesis of the petrographic and geochemical data, we suggest a provenance comprising basalts, tonalite–trondhjemite–granodiorite (TTG), and granite. Geochemical attributes indicate deposition of the detritus in an extensional backarc basin receiving sedimentary input from opposite directions. The opening and then closure of the South Delhi Basin was the last phase of the break-up of the supercontinent, columbia, which began by abortive rifting of the Udaipur belt and culminated in separation of the Aravalli–Bundelkhand–Dharwar block in the east and the East African orogen in the west.

Nanoparticles-protein interaction: Role in protein aggregation and clinical implications

This review helps to understand protein misfolding events, which results in protein aggregation, and hence to related neurodegenerative diseases. Many chaperones and folding factors are found inside the cell system for the proper folding of protein. If protein gets misfolded, it may accumulate in cells and can lead to several fatal diseases. In some cases, misfolded proteins aggregated in form of loop-sheet polymer and amyloid fibril when they escape the degradation process and leads to neurodegenerative disorders. Nanoparticles (NPs) are nano-sized materials, can be formulated by using organic molecules such as gelatin, chitosan, inorganic molecules, metals such as iron, gold, silver, etc. NPs unite with proteins and form a dynamic nanoparticle-protein (NP-P) corona. Conformational changes may be induced in adsorbed protein by this NP-P corona which might change overall bio-reactivity of NP. They can influence correct folding of unfolded or misfolded protein and prevent their aggregation which may be helpful in the cure of neurodegenerative disorders. Due to high area:size ratio, NPs have higher advantages over bulk materials. Hence, the effect of NPs on the proper protein folding opens new gateways to produce a biologically active three dimensional biomolecule.

How methyl cyanide induces aggregation in all-alpha proteins: A case study in four albumins

Serum albumins are chief carrier of ligands in blood, hence important in clinical biotechnology. The effects of methyl cyanide (MeCN), a chief solvent of reverse phase chromatography, on four mammalian serum albumins (human, bovine, porcine and rabbit sources) were studied at neutral pH with the help of scattering, circular dichroism, IR and fluorescence spectroscopy. We have detected an intermediate state in the presence of 20% (v/v) MeCN, having 8-9% higher alpha-helical structure than that of their native states. In the presence of 60% (v/v) MeCN another intermediate was observed with non-native beta-sheet structure and high tendency to form aggregates.

Native-like Tertiary Structure in the Mucor miehei Lipase Molten Globule State Obtained at Low pH

Studies on the acid‐induced denaturation of Mucor miehei lipase (E.C. 3.1.1.3) were performed by circular dichroism (CD) spectroscopy, fluorescence emission spectroscopy and binding of hydrophobic dye, 1‐anilino 8‐naphthalenesulfonic acid (ANS). Acid denaturation of the lipase showed loss of secondary structure and alterations in the tertiary structure in the pH range 4 to 2 and 7 to 2 respectively, suggesting that the lipase exists as an acid‐unfolded state ∼pH 2.0. A further decrease in pH (from 2.0 to 1.0) resulted in a second transition, which corresponded to the formation of both secondary and tertiary structures. The acid unfolded state at around pH 2.0 has been characterized by significant loss of secondary structure and a small increase in fluorescence intensity with a blue shift of 2 nm, indicating shift of tryptophan residues to less polar environment. Interestingly, the lipase at pH 1.0 exhibits characteristics of molten globule, such as enhanced binding of hydrophobic dye (ANS), native‐like secondary structure and slightly altered tryptophanyl environments. That the molten globule of the lipase at pH 1.0 also possesses native‐like tertiary structure is an interesting observation made for this lipase. IUBMB Life, 59: 179‐186, 2007

Harmful Effect of Detergents on Lipase

In order to study effects of detergents at molecular level, we have done activity measurements of wheat germ lipase in increasing concentration of some commercial detergents. Conformational changes in protein structure using circular dichroism and fluorescence spectroscopy were studied in increasing concentration of sodium dodecyl sulfate. Our study proves that detergents may lead to loss of enzymatic activity and structure of plant enzymes. Since detergents are common source of pollution in water bodies and the water from these resources can be used in fields, our study may prove helpful in creating awareness about harmful action of detergents.

A novel multicopper oxidase (laccase) from cyanobacteria: Purification, characterization with potential in the decolorization of anthraquinonic dye

A novel extracellular laccase enzyme produced from Spirulina platensis CFTRI was purified by ultrafiltration, cold acetone precipitation, anion exchange and size exclusion chromatography with 51.5% recovery and 5.8 purification fold. The purified laccase was a monomeric protein with molecular mass of ~66 kDa that was confirmed by zymogram analysis and peptide mass fingerprinting. The optimum pH and temperature of the enzyme activity was found at 3.0 and 30°C using ABTS as substrate but the enzyme was quite stable at high temperature and alkaline pH. The laccase activity was enhanced by Cu+2, Zn+2 and Mn+2. In addition, the dye decolorization potential of purified laccase was much higher in terms of extent as well as time. The purified laccase decolorized (96%) of anthraquinonic dye Reactive blue- 4 within 4 h and its biodegradation studies was monitored by UV visible spectra, FTIR and HPLC which concluded that cyanobacterial laccase can be efficiently used to decolorize synthetic dye and help in waste water treatment.