Characterization of pH-induced conformational changes in recombinant DENV NS2B-NS3pro.

The increasing frequency of Dengue is a cause of severe epidemics and therefore demands strategies for effective prevention, diagnosis, and treatment. DENV-protease is being investigated as a potential therapeutic target. However, due to the flat and highly charged active site of the DENV-protease, designing orthosteric medicines is very difficult. In this study, we have done a thorough analysis of pH-dependent conformational changes in recombinantly expressed DENV protease using various spectroscopic techniques. Our spectroscopic study of DENV protease (NS2B-NS3pro) at different pH conditions gives important insights into the dynamicity of structural conformation. At physiological pH, the DENV-protease exists in a random-coiled state. Lowering the pH promotes the formation of alpha-helical and beta-sheet structures i.e. gain of secondary structure as shown by Far-UV CD. The light scattering and Thioflavin T (ThT)-binding assay proved the aggregation-prone tendency of DENV-protease at pH 4.0. Further, the confocal microscopy image intensity showed the amorphous aggregate formation of DENV protease at pH 4.0. Thus, the DENV protease acquires different conformations with changes in pH conditions. Together, these results have the potential to facilitate the design of a conformation destabilizer-based therapeutic strategy for dengue fever.

Tuning the aggregation behavior of human insulin in the presence of luteolin: An in vitro and in silico approach.

Protein misfolding and related formation of amyloid fibrils are associated with several conformational diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), prion diseases, and Diabetes mellitus, Type 2 (DM-II). Several molecules including antibiotics, polyphenols, flavonoids, anthraquinones, and other small molecules are implicated to modulate amyloid assembly. The stabilization of the native forms of the polypeptides and prevention of their misfolding and aggregation are of clinical and biotechnological importance. Among the natural flavonoids, luteolin is of great importance because of its therapeutic role against neuroinflammation. Herein, we have explored the inhibitory effect of luteolin (LUT) on aggregation of a model protein, human insulin (HI). To understand the molecular mechanism of the inhibition of aggregation of HI by LUT, we employed molecular simulation, UV-Vis, fluorescence, and circular dichroism (CD) spectroscopies along with the dynamic light scattering (DLS). The analysis of the tuning of the HI aggregation process by luteolin revealed that interaction of HI with LUT resulted in the decrease in binding of the various fluorescent dyes, such as thioflavin T (ThT) and 8-anilinonaphthalene-1-sulfonic acid (ANS) to this protein. Retention of the native-like CD spectra and resistance to the aggregation in the presence of LUT has confirmed the aggregation inhibitory potential of LUT. The maximum inhibitory effect was found at the protein-to-drug ratio of 1:12, and no significant change was observed beyond this concentration.

Evaluating the inhibitory potential of natural compound luteolin on human lysozyme fibrillation.

Numerous pathophysiological conditions known as amyloidosis, have been connected to protein misfolding leading to aggregation of proteins. Inhibition of cytotoxic aggregates or disaggregation of the preformed fibrils is thus one of the important strategies in the prevention of such diseases. Growing interest and exploration of identification of small molecules mainly natural compounds can prevent or delay amyloid fibril formation. We examined the mechanism of interaction and inhibition of human lysozyme (HL) aggregates with luteolin (LT). Biophysical and computational approaches have been employed to study the effect of LT on HL amyloid aggregation. Transmission Electronic Microscopy, Thioflavin T fluorescence, UV-vis spectroscopy, and RLS demonstrates that LT inhibit HL fibril formation. ANS fluorescence and hemolytic assay was also employed to examine the effect of the LT on toxicity of HL aggregation. Docking and molecular dynamics results showed that LT interacted with HL via hydrophobic and hydrogen interactions, thus reducing fibrillation levels. These findings highlight the benefit of polyphenols as safe therapy for preventing amyloid related diseases.

Mechanistic insight into inhibition of amyloid fibrillation of human serum albumin by Vildagliptin.

Protein aggregation leads to several human pathologies such as Alzheimer's disease (AD), type 2 diabetes (T2D), Parkinson's disease (PD), etc. Due to the overlap in the mechanisms of type 2 diabetes and brain disorders, common effective pharmacological interventions to treat both T2D and AD is under extensive research. Therefore, major aim of research is to repurpose already established treatment of diabetes to cure AD as well. This study evaluates mechanistic insight into anti-amyloidogenic potential of anti-diabetic drug Vildagliptin (VLD) on human serum albumin fibrillation (HSA) by using biophysical, calorimetric, imaging techniques along with hemolytic assay. Dynamic light scattering (DLS) and Rayleigh light scattering (RLS) results showed presence of few small-sized aggregates in the presence of VLD which are formed by deaccelerating the amyloidogenesis as shown by thioflavin T (ThT) fluorescence and Congo red (CR) binding assay. Further, Isothermal titration calorimetry (ITC), steady state fluorescence quenching, molecular docking results revealed that VLD form complex with amyloid facilitating state of HSA and consequently mask the hydrophobic residues involved in amyloidogenesis as evident from decrease in ANS fluorescence. Differential scanning calorimetry (DSC) results confirm that VLD stabilizes the amyloid facilitating state of HSA. In addition, SEM images demonstrated that VLD alleviates the hemolytic effect induced by fibrils of HSA. This study reports VLD as a potential inhibitor of amyloid fibrillation and provides promising results to repurpose VLD as a drug candidate for the cure of Alzheimer's diseases along with diabetes.

Multifunctionality and intrinsic disorder of royal jelly proteome.

Royal Jelly (RJ) is a gelatinous white-yellowish fluid, possessing a sour taste and a slight phenolic smell that is secreted by the hypopharyngeal and mandibular salivary glands of the nurse honeybees, and is used in nutrition of larvae and adult queens. Similar to other substances associated with the activities of honeybees, RJ not only contains nutritive components, such as carbohydrates, proteins, peptides, lipids, vitamins, and mineral salts, but also represents a natural ingredient with cosmetic and health-promoting properties. RJ is characterized by remarkable multifunctionality, possessing numerous biological activities. Although this multifunctionality of RJ can be considered as a consequence of its complex nature, many proteins and peptides in RJ are polyfunctional entities themselves. In this article, we show that RJ proteins contain different levels of intrinsic disorder, have sites of post-translational modifications, can be found in multiple isoforms, and many of them possess disorder-based binding sites, suggesting that the conformational ensembles of the RJ proteins might undergo change as a result of their interaction with specific binding partners. All these observations suggest that the multifunctionality of proteins and peptides from RJ is determined by their structural heterogeneity and polymorphism, and serve as an illustration of the protein structure-function continuum concept.

Unravelling the inhibitory and cytoprotective potential of diuretics towards amyloid fibrillation.

Protein misfolding and deposition of aggregated proteins inside as well as outside of the cells have been associated with several neurotoxic and neurodegenerative disorders like Alzheimer's, Parkinson's and familial amyloid polyneuropathy etc. that could be controlled by anti-aggregation methodologies employing either inhibition or disaggregation of toxic aggregates. Also, the Alzheimer's disease develops in later life is somehow related to the high mid-life blood pressure. Therefore the present work targets the amyloid inhibiting potential of diuretics (a class of antihypertensive drugs) - Indapamide (INDP) and Hydrochlorothiazide (HCTZ) against human serum albumin (HSA) and human lysozyme (HL) fibrillogenesis. The effect of both INDP and HCTZ on the kinetics of amyloid formation of HSA and HL was illustrated and various biophysical techniques like Thioflavin T (ThT) and 8-Anilinonaphthalene-1-sulfonic acid (ANS) fluorescence measurement, Congo red measurements and circular dichroism (CD) measurements depicted the inhibitory action of both INDP and HCTZ in a dose dependent manner. Transmission Electronic Microscopy (TEM) confirmed the absence of fibrillar structures when HSA and HL were co-incubated with INDP and HCTZ. In addition, molecular docking results revealed that both the drugs interacts with HSA and HL through hydrophobic interactions as well as hydrogen bonding, and also showed non-hemolytic activity on human RBCs demonstrated by the Hemolytic assay. Thus, both INDP and HCTZ could be propitious as a therapeutic agent and aid in the cure of amyloid related diseases.

An interplay of structure and intrinsic disorder in the functionality of peptidylarginine deiminases, a family of key autoimmunity-related enzymes.

Citrullination is a post-translation modification of proteins, where the proteinaceous arginine residues are converted to non-coded citrulline residues. The immune tolerance to such citrullinated protein can be lost, leading to inflammatory and autoimmune diseases. Citrullination is a chemical reaction mediated by peptidylarginine deiminase enzymes (PADs), which are a family of calcium-dependent cysteine hydrolase enzymes that includes five isotypes: PAD1, PAD2, PAD3, PAD4, and PAD6. Each PAD has specific substrates and tissue distribution, where it modifies the arginine to produce a citrullinated protein with altered structure and function. All mammalian PADs have a sequence similarity of about 70-95%, whereas in humans, they are 50-55% homologous in their structure and amino acid sequences. Being calcium-dependent hydrolases, PADs are inactive under the physiological level of calcium, but could be activated due to distortions in calcium homeostasis, or when the cellular calcium levels are increased. In this article, we analyze some of the currently available data on the structural properties of human PADs, the mechanisms of their calcium-induced activation, and show that these proteins contain functionally important regions of intrinsic disorder. Citrullination represents an important trigger of multiple physiological and pathological processes, and as a result, PADs are recognized to play a number of important roles in autoimmune diseases, cancer, and neurodegeneration. Therefore, we also review the current state of the art in the development of PAD inhibitors with good potency and selectivity.

Elucidating the Inhibitory Potential of Designed Peptides Against Amyloid Fibrillation and Amyloid Associated Cytotoxicity.

Inhibition of fibrillation process and disaggregation of mature fibrils using small peptide are the promising remedial strategies to combat neurodegenerative diseases. However, designing peptide-based drugs to target ß-sheet-rich amyloid has been a major challenge. The current work describes, for the first time, the amyloid inhibitory potential of the two short peptides (selected on the basis of predisposition of their amino acid residues toward ß-sheet formation) using combination of biophysical, imaging methods, and docking approaches. Results showed that peptides employed different mechanisms to inhibit the amyloid fibrillation. Furthermore, they were also effective in blocking the amyloid fibrillation pathway. In contrary to the insulin fibrillar mesh, significantly less fibrillar species appeared in the presence of peptides, as confirmed by transmission electron microscopy. Circular dichroism analysis indicated that although peptides did not stabilize the native state of insulin, they inhibited amyloid aggregation by reducing the formation of ß-sheet rich structures. Hemolytic assay revealed the non-hemolytic nature of the species formed when insulin was co-incubated with the peptides. Therefore, despite the inherent potential to form ß-sheet structure, these peptides inhibited the amyloid formation and potentially can be used as therapeutics for the treatment of amyloid-related diseases.

Sauromatum guttatum lectin: Spectral studies, lectin-carbohydrate interaction, molecular cloning and in silico analysis.

Non-immune carbohydrate binding proteins are broadly defined as lectins. Having been reported from all kingdoms of life, phytolectins are the most widely studied group of lectins. Sauromatum guttatum agglutinin (SGA) was isolated from the plant tubers and characterized for structural variations due to solvent perturbation using polarimetry, fluorescence and light scattering. For the ß-sheet rich SGA, a pH and temperature induced molten globule like intermediate was identified. In isothermal titration microcalorimetry, SGA demonstrated cooperative binding to a complex glycoprotein in enthalpically driven mechanism. Fine sugar specificity exploration identified core pentasaccharide as the most common and highest binding motif with complex N-glycans and fucosylated core N-glycans as additional motifs. Molecular cloning of SGA which has previously been demonstrated to have anti-cancer and anti-insect activities is being reported for the first time. Full length cDNA sequence was obtained with RACE-PCR based upon the conserved carbohydrate recognition site [QXDXNXVXY] present in all GNA-related lectins. Quaternary structure was proposed by homology modeling and an attempt was made to explain the structure-function relationship by in silico analysis.

In Vitro Elucidation of the Folding Intermediates and Aggregate Formation of Hemoglobin Induced by Acetonitrile: A Multispectroscopic Approach.

Acetonitrile is a mild solvent, which induces ß-sheet conformation in proteins. The global conformational changes in Hb in the presence of ACN were studied using intrinsic fluorescence experiments, acrylamide quenching, ANS fluorescence measurements, soret absorbance spectroscopy, fourier transform infrared spectroscopy, circular dichroism, thioflavin T and congo red assay. Molecular docking showed the binding of hydrophobic residues of Hb to ACN. Hb exists as a partially unfolded intermediate state at 30% v/v ACN. Hb aggregates were obtained at 60% v/v ACN concentration, which were further confirmed by transmission electron microscopy.

Biogenic terbium oxide nanoparticles as the vanguard against osteosarcoma.

The synthesis of inner transition metal nanoparticles via an ecofriendly route is quite difficult. This study, for the first time, reports synthesis of terbium oxide nanoparticles using fungus, Fusarium oxysporum. The biocompatible terbium oxide nanoparticles (Tb2O3 NPs) were synthesized by incubating Tb4O7 with the biomass of fungus F. oxysporum. Multiple physical characterization techniques, such as UV-visible and photoluminescence spectroscopy, TEM, SAED, and zeta-potential were used to confirm the synthesis, purity, optical and surface characteristics, crystallinity, size, shape, distribution, and stability of the nanoemulsion of Tb2O3 NPs. The Tb2O3 NPs were found to inhibit the propagation of MG-63 and Saos-2 cell-lines (IC50 value of 0.102µg/mL) and remained non-toxic up to a concentration of 0.373µg/mL toward primary osteoblasts. Cell viability decreased in a concentration-dependent manner upon exposure to 10nm Tb2O3 NPs in the concentration range 0.023-0.373µg/mL. Cell toxicity was evaluated by observing changes in cell morphology, cell viability, oxidative stress parameters, and FACS analysis. Morphological examinations of cells revealed cell shrinkage, nuclear condensation, and formation of apoptotic bodies. The level of ROS within the cells-an indicator of oxidative stress was significantly increased. The induction of apoptosis at concentrations ≤IC50 was corroborated by 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) staining (DNA damage and nuclear fragmentation). Flow-cytometric studies indicated that the response was dose dependent with a threshold effect.

Comparative insight into surfactants mediated amyloidogenesis of lysozyme.

Electrostatic and hydrophobic interactions have an important role in the protein aggregation. In this study, we have investigated the effect of charge and hydrophobicity of oppositely charged surfactants i.e., anionic (AOT and SDS) and cationic (CTAB and DTAB) on hen egg white lysozyme at pH 9.0 and 13.0, respectively. We have employed various methods such as turbidity measurements, Rayleigh light scattering, ThT, Congo red and ANS dye binding assays, far-UV CD, atomic force microscopy, transmission electron and fluorescence microscopy. At lower molar ratio, both anionic and cationic surfactants promote amyloid fibril formation in lysozyme at pH 9.0 and 13.0, respectively. The aggregation was proportionally increased with respect to protein concentration and hydrophobicity of surfactant. The morphology of aggregates at both the pH was fibrillar in structure, as visualized by dye binding and microscopic imaging techniques. Initially, the interaction between surfactants and lysozyme was electrostatic and then hydrophobic as investigated by ITC. This study demonstrates the crucial role of charge and hydrophobicity during amyloid fibril formation.

Structural variations and molten globule state in Arisaema helliborifolium lectin under various treatments as monitored by spectroscopy.

Solvent perturbation was used to study variations in structure of Arisaema helliborifolium lectin (AHL) with the help of circular dichroism (CD), intrinsic fluorescence (IF), extrinsic fluorescence, quenching and dynamic light scattering (DLS). AHL was studied under acidic, alkaline and 6 M guanidine hydrochloride (GuHCl) equilibrium states. Three structural states were identified for AHL at different conditions, that are native (N; pH 7.0), molten globule (MG; pH 2.0) and unfolded (U; pH 12.0). CD analysis revealed that 50% of secondary structure of AHL was ß-sheet component. A complete loss of secondary structure was observed at GuHCl treatment. The tertiary structural changes as studied by changes in microenvironment of trp residues also suggested a pH induced MG state as in case of CD. Parameter-A analysis pointed at the multi-step unfolding process of lectin under varying pH (pH 1-13). A comparision of CD and IF data further indicated that different pathways were followed for secondary and tertiary structure unfolding. Tryptophans of native AHL were only partially exposed to solvent belonging to Class II. Hydrodynamic diameter (Dh ) measurements of AHL via DLS also confirmed of a pH induced molten globule. A thermally induced molten globule was identified for AHL between 54-60 °C as monitored by DLS. An irreversible thermal denaturation was observed with the formation of a large aggregate. The Dh of AHL at neutral pH was confirmed by transmission electron microscopy (TEM).

Conformational transitions induced by in vitro macromolecular crowding lead to the amyloidogenesis of buffalo heart cystatin.

The biological cells and extracellular matrix exhibit a highly crowded environment, called as macromolecular crowding. Crowding significantly influences protein structure and may lead to its aggregation. In the present study, buffalo heart cystatin (BHC), after purification from buffalo heart tissue, has been used as a model protein for studying effect of macromolecular crowding in the presence of high concentrations of bovine serum albumin (BSA), poly-ethylene glycol-1000 (PEG-1000), and poly-ethylene glycol-4000 (PEG-4000). Cystatins are thiol protease inhibitors and found to be involved in various important physiological processes. Functional inactivation of BHC was observed upon crowding, which varied as a function of concentration and molecular weight of crowding agents as well as incubation time. Structural changes of BHC at tertiary and secondary level were detected with the help of fluorescence and CD spectroscopy. CD analysis showed changes of α-helix to ß-sheet, which could be due to aggregation. The ANS-fluorescence study suggested the unfolding and presence of some partially folded intermediates. Increase in ThT-fluorescence and absorption of Congo red spectra with red shift, confirmed the amyloid type aggregation of BHC in the presence of various crowding agents. Finally, electron microscopy provided the physical evidence about the formation of amyloid fibrils. Results suggested that among the various crowding agents used, amyloidogenesis of BHC was maximal in case of BSA followed by PEG-4000 and least for PEG-1000. The present work makes an important contribution in crowding mediated protein aggregation, which can have implications of potential interest.

pH-dependent differential interacting mechanisms of sodium dodecyl sulfate with bovine serum fetuin: a biophysical insight.

Sodium dodecyl sulfate (SDS)-glycoprotein interaction serves as a model for a biological membrane. To get mechanistic insight into the interaction of SDS and glycoprotein, the effect of SDS on bovine serum fetuin (BSF) was studied in subcritical micellar concentrations at pH 7.4 and pH 2 using multiple approaches. SDS interacts electrostatically with BSF through its negatively charged head groups at pH 2 and hydrophobically via its alkyl chains at pH 7.4 up to a 1:20 molar ratio of BSF to SDS. However, at higher concentrations of SDS, BSF undergoes amyloid fibril formation at pH 2, as confirmed by enhanced ThT fluorescence, ß-sheet formation, and TEM microscopy, whereas BSF undergoes induction of an α-helical structure in the presence of higher SDS concentration at pH 7.4. The increase in α-helical content with increasing SDS concentrations constrains the environment around tryptophan. As a consequence, the interconversion of tryptophan conformers decreases, resulting in a decrement of the fluorescence lifetime for BSF in the presence of SDS at pH 7.4.

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.

Protonation favors aggregation of lysozyme with SDS.

Different proteins have different amino acid sequences as well as conformations, and therefore different propensities to aggregate. Electrostatic interactions have an important role in the aggregation of proteins as revealed by our previous report (J. M. Khan et al., PLoS One, 2012, 7, e29694). In this study, we designed and executed experiments to gain knowledge of the role of charge variations on proteins during the events of protein aggregation with lysozyme as a model protein. To impart positive and negative charges to proteins, we incubated lysozyme at different pH values of below and above the pI (∼11). Negatively charged SDS was used to 'antagonize' positive charges on lysozyme. We examined the effects of pH variations on SDS-induced amyloid fibril formation by lysozyme using methods such as far-UV circular dichroism, Rayleigh scattering, turbidity measurements, dye binding assays and dynamic light scattering. We found that sub-micellar concentrations of SDS (0.1 to 0.6 mM) induced amyloid fibril formation by lysozyme in the pH range of 10.0-1.0 and maximum aggregation was observed at pH 1.0. The morphology of aggregates was fibrillar in structure, as visualized by transmission electron microscopy. Isothermal titration calorimetry studies demonstrated that fibril formation is exothermic. To the best of our current understanding of the mechanism of aggregation, this study demonstrates the crucial role of electrostatic interactions during amyloid fibril formation. The model proposed here will help in designing molecules that can prevent or reverse the amyloid fibril formation or the aggregation.

Hydrophobicity alone can not trigger aggregation in protonated mammalian serum albumins.

Amyloid fibrils are associated with neurodegenerative disorders and are formed by a number of proteins. In this study, the amyloid-forming behavior of several different serum albumins was examined at pH 3.5 i.e., about two pH units below their isoelectric points (pI ∼ 5.5) to examine the roles played by negative charge and hydrophobicity of exogenously added surfactants such as SDS, SDBS and AOT. The propensities of SDS, SDBS and AOT to promote the formation of amyloid fibrils were investigated by using measurements of turbidity, Rayleigh scattering, ThT and CR dye binding, DLS as well as far-UV CD. At submicellar concentrations of SDS and SDBS (0.5-2.5 mM) amyloid fibrils were formed by all albumins studied whereas at higher concentrations amyloid fibril formation was completely inhibited. Interestingly AOT promoted amyloid fibril formation up to 11 mM without any inhibition. The interaction between the albumins and the surfactants was exothermic, as confirmed by isothermal titration calorimetry (ITC). From the turbidity, Rayleigh scattering and dynamic light scattering data, it was concluded that amyloid induction was promoted most by AOT followed by SDBS and SDS. Similar studies were performed at pH 7.4 i.e., about two units of pH above the albumins pI, and no amyloid fibrils were formed. From these studies we conclude that negatively charged surfactants induce amyloid fibril formation in serum albumins with the help of electrostatic and hydrophobic interactions. Besides the study performed at pH 7.4 indicates that hydrophobic interactions alone can not induce aggregation in serum albumins.

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 ß-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 ß-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 ß-sheet usually alters the normal protein to amyloidogenic resulting in a variety of protein conformational disorders such as diabetes, prion and Huntington's.

Monomeric banana lectin at acidic pH overrules conformational stability of its native dimeric form.

Banana lectin (BL) is a homodimeric protein categorized among jacalin-related family of lectins. The effect of acidic pH was examined on conformational stability of BL by using circular dichroism, intrinsic fluorescence, 1-anilino-8-napthalene sulfonate (ANS) binding, size exclusion chromatography (SEC) and dynamic light scattering (DLS). During acid denaturation of BL, the monomerization of native dimeric protein was found at pH 2.0. The elution profile from SEC showed two different peaks (59.65 ml & 87.98 ml) at pH 2.0 while single peak (61.45 ml) at pH 7.4. The hydrodynamic radii (R h) of native BL was 2.9 nm while at pH 2.0 two species were found with R h of 1.7 and 3.7 nm. Furthermore at, pH 2.0 the secondary structures of BL remained unaltered while tertiary structure was significantly disrupted with the exposure of hydrophobic clusters confirming the existence of molten globule like state. The unfolding of BL with different subunit status was further evaluated by urea and temperature mediated denaturation to check their stability. As inferred from high Cm and ΔG values, the monomeric form of BL offers more resistance towards chemical denaturation than the native dimeric form. Besides, dimeric BL exhibited a Tm of 77°C while no loss in secondary structures was observed in monomers even up to 95°C. To the best of our knowledge, this is the first report on monomeric subunit of lectins showing more stability against denaturants than its native dimeric state.