Umesh Chandra Halder

Tryptophan dynamics in the exploration of micro-conformational changes of refolded β-lactoglobulin after thermal exposure: A steady state and time-resolved fluorescence approach

Refolding intermediates of proteins, including molten globules, are likely to undergo dynamic conformational transitions. In this work, thermal unfolding and refolding of bovine β-lactoglobulin (β-lg) have been revisited to encounter such intermediate states. Lower thermal range (< 80°C) was selected to avoid irreversible aggregate formation. The gross kinetic refolding as monitored with the fluorophore, Trp19, was likely to be reversible but alteration in time resolved fluorescence parameters ruled out the possibility of micro-structural reversibility for the refolded partner. Time resolved fluorescence showed that the refolded protein still lacks some intact native conformation. Far-UV CD signals lack the signature of any secondary structural distortion in global structural context whereas near-UV CD signals were strongly indicative of perturbation in micro-structure surrounding the aromatic moieties which hardly revives after cooling. Steady state anisotropy results showed successfully the break-down of dimer to monomer form of β-lg within 50°C temperature range and augmentation in anisotropy up on further thermal stress reflected the reorganization of tryptophan residues into more restricted and rigid micro-environment as well as irreversible disulfide-linked dimer formation. Reliability of conformational reversibility in the thermal unfolding-refolding is still enigmatic on micro and global structural perspectives. Intermediate state prior to the completion of refolding of thermally exposed β-lg was identified through fluorescence studies.

Amyloid fibril formation by β-lactoglobulin is inhibited by gold nanoparticles

The endogenous deposition of protein fibrillar aggregates in the tissues is the cause of several neurodisorders. In the present study the native β-lactoglobulin (β-lg) has been driven towards amyloid fibrillar aggregates when it was exposed to heat at 75°C for 1h at pH 7.5. The citrate stabilized gold nanoparticle (AuNPs) of 20nm diameter is shown to inhibit the thermal aggregation of β-lg. The stability of the β-lg against heat stress was assessed by studying its aggregation at 75°C, either in presence or in absence of AuNPs. AuNPs stabilizes the monomeric and dimeric forms of the β-lg inhibiting the nucleation and elongation for the formation of higher aggregates. Incubation of β-lg with AuNPs at 75°C results in the formation of smaller ragged aggregates. Results show that AuNPs possess the capability of inhibiting the formation of amyloid fibrillar aggregates of β-lg in a concentration-dependent manner and may thus facilitate the refolding into native like structure. AuNPs thus serve as nano-chaperon to inhibit the protein aggregation. Thus chaperon like activity of AuNP may be exploited in the design of rational therapeutics for the neurodegenerative diseases.

Characterization of Trichoderma reesei endoglucanase ii expressed heterologously in Pichia pastoris for better biofinishing and biostoning

The endoglucanase II of Trichoderma reesei is considered the most effective enzyme for biofinishing cotton fabrics and biostoning denim garments. However, the commercially available preparation of endoglucanase II is usually mixed with other cellulase components, especially endoglucanase I, resulting in hydrolysis and weight loss of garments during biofinishing and biostoning. We thus isolated the endoglucanase II gene from T. reesei to express this in Pichia pastoris, under the control of a methanol-inducible AOX1 promoter, to avoid the presence of other cellulase components. A highly expressible Mut+ transformant was selected and its expression in BMMH medium was found most suitable for the production of large amounts of the recombinant protein. Recombinant endoglucanase II was purified to electrophoretic homogeneity, and functionally characterized by activity staining. The specific activity of recombinant endoglucanase II was found to be 220.57 EU/mg of protein. Purified recombinant endoglucanase II was estimated to have a molecular mass of 52.8 kDa. The increase in molecular mass was likely due to hyperglycosylation. Hyperglycosylation of recombinant endoglucanase II secreted by P. pastoris did not change the temperature or pH optima as compared to the native protein, but did result in increased thermostability. Kinetic analysis showed that recombinant endoglucanase was most active against amorphous cellulose, such as carboxymethyl cellulose, for which it also had a high affinity.

The Role of Neutrophil Estrogen Receptor Status on Maspin Synthesis via Nitric Oxide Production in Human Breast Cancer

Purpose Estrogen, through its binding to nuclear estrogen receptor (ER), has been implicated in the development of human breast cancer. The presence or absence of ER in breast lesions has been used to classify breast cancer into ER+ or ER- type. Maspin, an anti-breast cancer protein produced in normal mammary cells, has also been reported to control the condition. Studies have been conducted to determine the role of ER+ and ER- status in neutrophils in the synthesis of maspin in human breast cancer. Methods Maspin presence was determined by enzyme linked immunosorbent assay, while nitric oxide (NO) level was determined using the methemoglobin method. Results Scatchard plots of the equilibrium binding of estrogen demonstrated the presence of 4.18×107 receptors per normal neutrophil and 2.46×107 receptors per ER+ neutrophil with a similar dissociation constant (0.926 nM). The ER- type showed nonspecific estrogen binding only. At 0.6 nM estrogen, NO synthesis was maximally increased to 1.829 and 0.887 µM NO/109 cells at 4 hours in normal and ER+ neutrophils respectively, with synthesis of 2.383 and 1.422 nM maspin in normal and ER+ neutrophils respectively. Estrogen failed to produce these effects in ER- neutrophils. Conclusion ER status in neutrophils determined maspin synthesis in breast cancer through the stimulation of NO synthesis. Neutrophils with ER- status which do not produce any maspin when treated with estrogen, might imply a worse prognostic outcome in ER- breast cancer due to the lack of anti-breast cancer protein synthesis.

Unfolding diminishes fluorescence resonance energy transfer (FRET) of lysine modified β-lactoglobulin: Relevance towards anti-HIV binding

In accordance with the recent reports by Ng et al. (2001) [12] and Pellengrini et al. (2001) [13], that acetylated and succinylated β-lg has a potent HIV-I and HIV-II type enzyme inhibitory activity, a spectro-fluoremetric approach has been made to understand the mode of interactions playing the key role in inhibition process. In this article, interactions between lysine modified bovine β-lactoglobulin (β-lg) and a hydrophobic fluorescence probe, 1-anilinonapthalene-8-sulfonate (ANS), have been studied with the help of fluorescence resonance energy transfer (FRET) process. Lysine residues of β-lg were modified by acetylation and succinylation. Tryptophan-19 of intact β-lg efficiently transfers energy to ANS, whereas in derivatives, it unexpectedly failed to promote energy transfer in spite of being more solvent exposed with an appreciable overlap integral. Efficient fluorescence resonance energy transfer (FRET) is a consequence of good overlap between emission and absorption spectra of donor and acceptor respectively. Therefore, linearity of this relationship becomes questionable in case of modified bio-molecules. Furthermore, time resolved studies showed that in the derivatives, hydrophobic cavities of β-lg were collapsed so that ANS failed to recognize the deep interior pockets leading to the loss of longer lifetime component. Modifications also prohibited the ionic association through surface leading to the loss of shorter lifetime component. Hence, chemical modification destabilizes β-lg conformations that affect FRET and interactions are strictly electrostatic.

Multispectroscopic analysis and molecular modeling to investigate the binding of beta lactoglobulin with curcumin derivatives

Bovine beta lactoglobulin (β-lg), the major whey protein, has a great affinity for a wide range of organic compounds like fatty acids, retinol etc. Curcumin, a polyphenolic antioxidant present in turmeric and its isoxazole (IOC) and pyrazole (PY) derivatives have been elicited worldwide for their therapeutic activities. However, the nature of interaction of β-lg with these derivatives remains unexplored. Fluorescence quenching studies suggest a static quenching mechanism for both the compounds. The average distances of 7.28 nm and 7.33 nm have been determined for IOC and PY respectively for energy transfer based on FRET which have application in many biological and biophysical fields. Circular dichroism spectra (CD) and Fourier transform infrared spectroscopy (FTIR) have been utilized to analyze the influence on the secondary structure of the protein. Docking simulation reveals a possible mechanism for different quenching behaviours and modes of binding preferred by the two compounds. Our findings will be helpful in the design of the drugs and other biologically active molecules that bind more strongly to β-lg and have the ability to show FRET.

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.

Curcumin inhibits the Al(III) and Zn(II) induced amyloid fibrillation of β-lactoglobulin in vitro

Accumulation of ordered protein aggregates (or amyloids) is responsible for several neurodegenerative diseases. The behaviour of amyloidal fibril formation of β-lactoglobulin (β-lg) during heat treatment depends on the environmental conditions. In this study the Al(III) and Zn(II) induced amyloid fibrillations of β-lg, in the absence and presence of curcumin, were evaluated using fluorescence, Thioflavin T, Congo red, Rayleigh scattering, dynamic light scattering analysis, FT-IR, CD spectroscopy and transmission electron microscopy. Curcumin, a natural phenolic antioxidant, is capable of binding with Al3+, Zn2+ and β-lg. Our experimental findings demonstrate that the metal–curcumin mixture can inhibit the transition from less structured oligomers to β-sheet rich protofibrils which act as seeding factors for further fibrillization. The Al(III)–curcumin mixture has greater inhibition capability than the Zn(II)–curcumin mixture of heat treated metal induced aggregation of β-lg.