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β-Glucan, a complex polysaccharide derived from fungal and yeast cell walls, plays a crucial role in modulating immune responses through their interaction with receptors such as Dectin-1 and Complement receptor 3 (CR-3). This review provides an in-depth analysis of the molecular mechanisms by which β-glucans activate receptor-mediated signalling pathways, focusing particularly on the LC3-associated phagocytosis (LAP) and autophagy pathways. Hence, we explore how β-glucan receptor engagement stimulates NADPH oxidase 2 (NOX-2), leading to the intracellular production of significant level of reactive oxygen species (ROS) essential for both conventional autophagy and LAP. While significant progress has been made in elucidation of downstream signaling by glucans, the regulation of phago-lysosomal maturation and antigen presentation during LAP induction still remains less explored. This review aims to provide a comprehensive overview of these pathways and their regulation by β-glucans. By consolidating the current knowledge, we seek to highlight how these mechanisms can be leveraged for therapeutic applications, particularly in the context of tuberculosis (TB) management, where β-glucans could serve as host-directed adjuvant therapies to combat drug-resistant strains. Despite major advancements in this field, currently key research gaps still persist, including detailed molecular interactions between β-glucan receptors and NOX-2 and the translation of these findings to in-vivo models and clinical investigations. This review underscores the need for further research to explore the therapeutic potential of β-glucans in managing not only tuberculosis but also other diseases such as cancer, cardiovascular conditions, and metabolic disorders., Competing Interests: Declaration of competing interest All authors have reviewed and approved the manuscript for submission to this journal. The authors declare that there are no conflicts of interest related to this article., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Tuberculosis (TB), attributable to Mycobacterium tuberculosis (M.tb.), constitutes a formidable global health challenge, particularly with the proliferation of multidrug-resistant (MDR-TB) strains. The efficacious clearance of M.tb. from host cells is imperative for mitigating infection and averting disease progression. Autophagy, an intricate cellular mechanism for degrading and recycling biomolecules, plays a pivotal role in the immune response to M.tb. by facilitating the degradation of intracellular pathogen through the formation of autophagosomes and their subsequent fusion with lysosomes. The present study elucidates the therapeutic efficacy of Rifabutin loaded YDGP (DYDGP) microparticles within M.tb.infected macrophage. Our results show that the administration of DYDGP improve the membrane integrity of macrophage infected with H37Rv as well as MDR strains, as compared to that of untreated controls at 30 min, 6 h and 24 h post-exposure time points. DCFHDA staining elucidated that DYDGP treatment significantly enhances intracellular reactive oxygen species (ROS) production compared to blank YDGP, even in the presence of NOX-2 inhibitors. Furthermore, DYDGP promotes the biogenesis of acidic vesicular organelles and phago-lysosomal maturation, as corroborated by acridine orange and Lysotracker Red staining. Immunofluorescence and dansylcadaverine dual staining data evidenced that DYDGP treatment enhances autophagosome formation, autophagy induction and LC3 puncta formation within M.tb. infected macrophage at both 30 min and 24 h post-exposure time points. Further, protein expression analyses demonstrated that DYDGP treatment enhances the expression levels of NOX-2 and LC3, thereby confirming autophagy induction within M.tb. infected macrophage. Antimycobacterial efficacy assessments revealed that DYDGP treatment engendered significant reductions in colony-forming units (CFUs) of H37Rv (64, 40, 19), MDR32420 (44, 35, 18), MDR32422 (44, 39, 21), and MDR32521 (38, 22, 18) after 30 min, 24 h, and 48 h, exposure respectively. These findings accentuate DYDGP's potential to substantially attenuate M.tb. burden, including the addressal of MDR strains, thereby positioning it as a promising adjunctive therapy for augmenting TB treatment., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Bio-wastes are organic materials achieved through biological sources. The rice crop produces a substantial amount of biowaste in the form of rice husk, which is rich in cellulose. In this research, cellulose was extracted from rice husk by alkalization and bleaching process. The rice husk extracted cellulose was further used to develop cellulose hydrogel by using the sol-gel technique. The nonwoven fabric of industrial cotton waste was developed in three different GSM (50, 100, and 150). The nonwoven fabric was incorporated in the cellulose hydrogel having three different concentrations (1 %, 2 %, and 3 %) to develop the hydrogel non-woven cotton fabric composite for sustainable wound dressing applications. Moreover, prepared rice husk extracted cellulose hydrogel loaded with AgNO 3 (0.5 %, 1 %, and 1.5 %) for achieving antibacterial characteristics. The Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM) were employed to confirm the existence of cellulose hydrogel layers within the cotton nonwoven composite. The developed hydrogel S12 exhibited a maximum fluid absorbency of 1281.84 % with a tensile strength of 28.6 N and elongation of 40.96 %. The results show successful rice husk extracted cellulose hydrogel formation, exhibiting structural stability, excellent exudate absorbency and moisture management, antimicrobial efficacy, and sustainability., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Banana fibers are a sustainable material with natural mechanical strength and antibacterial properties. These fibers are extracted from the large amount of waste produced by banana pseudo stems annually. However, despite their numerous advantages, their stiffness and rough texture impede their full use in the textile. This research investigates the degumming treatment of banana fibers using enzyme combination and chemical methods to achieve spinnable soft banana fibers. An L9 orthogonal array was used in a Taguchi design of the experiment to optimize the process parameters. For enzyme combination degumming, the experimental setup comprised different quantities of hemicellulase, laccase, amylase, and pectinase; for chemical degumming, varied amounts of sodium hydroxide (NaOH) were used. The results indicate that enzyme-based degumming procedures produce better results than chemical treatments. Optimum enzyme combinations for various fiber qualities were found using the Taguchi design of experiments. These combinations included Hemicellulase 5 %, Laccase 5 %, Amylase 3 %, and Hemicellulase 5 %, Laccase 3 %, Pectinase 5 %. Without degrading the cellulose structure, these ideal enzyme combinations produced fibers with lower lignin content and higher cellulose percentages, moisture content, and tenacity values. By determining the most efficient enzyme combinations and their effects on fiber qualities, the study offers sustainable fiber processing methods for textile grade banana fiber., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

SARS-CoV-2 infection results in cytokine burst, leading to proinflammatory responses in lungs of COVID-19 patients. SARS-CoV-2 ORF3a triggers the generation of proinflammatory cytokines. However, the underlying mechanism of dysregulation of proinflammatory responses is not well understood. We studied the role of microRNA in the generation of proinflammatory responses as a bystander effect of SARS-CoV-2 ORF3a in human lung epithelial cells. We observed upregulation of hsa-miR-155-5p in SARS-CoV-2 ORF3a transfected human lung epithelial cells, which led to the reduced expression of SHIP1. This resulted in phosphorylation of AKT and NF-κB, which further led to the increased expression of the proinflammatory cytokines IL-6 and TNF-α. Additionally, overexpression and knockdown studies of hsa-miR-155-5p were performed to confirm the role of hsa-miR-155-5p in the regulation of the SHIP1. We demonstrated that hsa-miR-155-5p modulates the proinflammatory response by activating the PI3K/AKT pathway through the inhibition of SHIP1 in SARS-CoV-2 ORF3a transfected human lung epithelial cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The present study aims to develop and characterize biodegradable packaging films from lemon peel-derived pectin and chitosan incorporated with a bioactive extract from neem leaves. The films (PCNE) contained varying concentrations of neem leaf extract and were comprehensively assessed for their physical, optical, mechanical, and antimicrobial attributes. The thickness, moisture content, water solubility, and water vapor permeability of the biodegradable packaging films increased with the increasing concentration of neem leaf extract. Comparatively, the tensile strength of the films decreased by 42.05 % compared to the control film. The Scanning Electron Microscopy (SEM) confirmed that the resultant blended pectin-chitosan films showed a uniform structure without cracks. Furthermore, the analysis targeting Staphylococcus aureus and Aspergillus niger indicated that the films had potent antimicrobial activity. Based on these results, the optimum films were selected and subsequently applied on apricot fruits to increase their shelf life at ambient temperature. The findings, after examining factors such as colour, firmness, total soluble solids, shrinkage, weight loss, and appearance, concluded that the apricots coated by PCNE-5 had the most delayed signs of spoilage and increased their shelf life by 50 %. The results showed the potential applicability of lemon peel pectin-chitosan-neem leaf extract blend films in biodegradable food packaging., Competing Interests: Declaration of competing interest The authors declare that they do not have any conflict of interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Nanocoatings are ultra-thin layers on the nanoscale (<100 nm) that are deposited on the substrate to improve their properties and functionality. These nanocoatings provide significant advantages compared to traditional coating, including stain resistance, antimicrobial and antioxidant activities, odor control and delivery of active agents, and liquid repellence properties. In the food industry, nanocoating is widely used in the food packaging sector. In this regard, nanocoating offers antimicrobials and antioxidant properties to active food packaging by incorporating active bioactive compounds into materials used in already existing packaging. The application of nanocoating is applied to these kinds of food packaging with nano coating to improve shelf life, safety, and quality of food packaging. In smart/intelligent packaging, the active packaging coating is promising food packaging, which is designed by releasing preservatives and nanocoating as an antimicrobial, antifungal, antioxidant, barrier coating, and self-cleaning food contact surfaces. In addition, nanocoating can be used for food contact surfaces, kitchen utensils, and food processing equipment to create antimicrobial, antireflective, and dirt-repellent properties. These are critical properties for food processing, especially for meat and dairy processing facilities, which can reduce biofilm formation and prevent cross-contamination. Recently, appreciable growth in the development of the application of nanocoating as edible films for coating food products has emerged to improve food safety issues. In this regard, much scientific research in the area of nanocoating fruits and vegetables, and other food products was performed to address food safety issues. Hence, this promising technology can be a great addition to the agricultural and food industries. Thus, this review addresses the most relevant information about this technology and the applications of nanocoating in the food industry., Competing Interests: Declaration of competing interest The authors declare that they do not have any conflict of interests., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Alginate hydrogel is highly efficient for water filtration due to its anti-fouling nature and formation of strong hydration membranes. However, poor mechanical properties of alginate hydrogel membrane limit its installation in water treatment. There is a need to enhance mechanical properties of alginate hydrogel membranes using eco-friendly, cost-effective materials and technologies. In this work, hydroentangled non-woven from cotton waste (comber noil) fibers was prepared. This non-woven was immersed in solution of sodium alginate (0.5 %, 1 %, 1.5 %) followed by dipping in calcium chloride solution which resulted in gel formation on and into cotton fibers. The successful formation of gel on non-woven fabric was confirmed through FTIR (Fourier transform infrared spectroscopy) and properties of this composite membrane were analyzed by SEM (Scanning electron microscopy), XRD (X-ray diffraction), DSC (Differential scanning calorimeter), water contact, water flux, oil-water filtration, air permeability, tensile strength, and porosity tests. The results showed that porosity of prepared hydrogel membranes decreased with increasing alginate concentration from 0.5 % to 1.5 % which resulted in decreased water permeation flux from 2655 h -1 /m 2 to 475 h -1 /m 2 . The prepared membrane has separation efficiencies for the oil-water mixture in the range of 97.5 % to 99.5 %. Moreover, the developed samples also showed significant antibacterial activity as well as improved mechanical properties. The strength of the prepared membrane is in the range of 40 N to 80 N. The developed sodium alginate hydrogel-based non-woven membrane could have potential applications for commercial water filtration systems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Hydrogels which become increasingly important in the biomedical field are composed of a three-dimensional hydrophilic network. Pure hydrogels are usually weak and brittle; therefore, reinforcements are assimilated into the hydrogel structure to improve the mechanical strength of the hydrogels. However, even if mechanical properties are enhanced, drapability remains an issue. In that regard, natural fiber-reinforced composite hydrogel fibers for wound dressing application are investigated in this study. Kapok and hemp fibers were used as reinforcement to improve the strength of hydrogel fibers. The properties of the prepared composite hydrogel fibers were studied with Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and differential scanning calorimeter (DSC). The effect of alginate concentration and fiber weight percent on the mechanical characteristics and water absorbency was studied. Diclofenac sodium drug was loaded in the hydrogel fibers and investigated the drug release as well as antibacterial characteristics. Both fibers' reinforcement enhanced the strength of the alginate hydrogel fiber, but hemp reinforcement showed better mechanical properties. Kapok reinforcement resulted in a maximum tensile strength of 174 cN (1.24 % elongation) and 432 % exudate absorbency, while hemp reinforcement resulted in 185 cN (1.48 % elongation) and 435 % exudate absorbency. Statistical analysis revealed significant effects of sodium alginate concentration on tensile strength (p-value 0.042) and exudate absorbency (p-value 0.020) and of reinforcement (wt%) on exudate absorbency (p-value 0.043). Therefore, these composite hydrogel fibers with improved mechanical properties are capable of drug release and exhibit antibacterial effectiveness, making them a promising option for use as wound dressings., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Increasing the temperature by just a few degrees may lead to structural perturbation or unfolding of the protein and consequent loss of function. The concepts of flexibility and rigidity are fundamental for understanding the relationships between function, structure and stability. Protein unfolding can often be triggered by thermal fluctuations with flexible residues usually on the protein surface. Therefore, identification and knowledge of the effect of modification to flexible regions in protein structures are required for efficient protein engineering and the rational design of thermally stable proteins. The most flexible regions in protein are loops, hence their rigidification is one of the effective strategies for increasing thermal stability. Directed evolution or rational design by computational prediction can also lead to the generation of thermally stable proteins. Computational protein design has been improved significantly in recent years and has successfully produced de novo stable backbone structures with optimized sequences and functions. This review discusses intramolecular and intermolecular interactions that determine the protein structure, and the strategies utilized in the mutagenesis of mesophilic proteins to stabilize and improve the functional characteristics of biocatalysts by describing efficient techniques and strategies to rigidify flexible loops at appropriate positions in the structure of the protein., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The unique pool of features makes bacterial cellulose (BC) a robust platform to tailor its functionalities. Herein, the BC matrix was reinforced with multiwalled carbon nanotubes (MWCNT) to control infection and accelerate the healing process of diabetic wounds. The prepared BC-MWCNT composite film was characterized and antibacterial activity was assessed. Further, the in-vivo wound healing activity was performed and temporal expression of interleukin (IL-1α), tumor necrosis factor (TNF-α), vascular endothelial growth factor (VEGF) and platelets derived growth factor (PDGF) was quantitatively measured by real-time PCR. The characterization results confirmed the reinforcement of the BC matrix with MWCNT. The composite film showed antibacterial activity against all the tested strains. Moreover, the macroscopic analysis of the wound demonstrated faster closure of the diabetic wound in BC-MWCNT group (99% healing) as compared to negative control (77%) in 21 days. Histological studies further supported the results where complete reepithelization of the epidermis and healthy granulation tissue were observed in BC-MWCNT treated group. Molecular studies revealed that BC-MWCNT group showed relatively lesser expression of pro-inflammatory cytokines IL-1α and TNF-α and higher expression of VEGF than control that may have favored the faster healing. This study suggested that the tailorable properties of BC can be exploited to develop composites with potential applications in diabetic wound healing., (Copyright © 2022. Published by Elsevier B.V.)

Macromolecular crowding can have significant consequences on the structure and dynamics of a protein. The size and shape of a co-solute molecule and the nature of protein contribute significantly in macromolecular crowding, which results in different outcomes in similar conditions. The structure of apo-myoglobin (apo-Mb) both in the absence and presence of denaturants (GdmCl and urea) was investigated in crowded conditions at pH 7.0, with a comparable size of crowders (~70 kDa) but of different shapes (ficoll and dextran) at various concentrations using spectroscopic techniques like absorption and circular dichroism to monitor changes in secondary and tertiary structure, respectively. The crowders in the absence of denaturants showed structural stabilization of the tertiary structure while no significant change in the secondary structure was observed. The effect of crowders on the stability of the protein was also investigated using probes such as Δε 291 and θ 222 using chemical denaturants. The analysis of chemical-induced denaturation curves showed that both the crowders stabilize apo-Mb by increasing the values of the midpoint of transition (C m ) and change in free energy in the absence of denaturant (∆G D °), and it was observed that dextran 70 shows more stabilization than ficoll 70 under similar conditions. In this study apo-Mb showed stabilization under crowded conditions, which is a deviation from earlier work from our group where holo form of the same protein was destabilized. This study emphasizes that volume exclusion is a dominant force in a simple protein while soft interactions may play important role in the proteins that are possessing prosthetic group. Hence, the effect of crowders is protein-dependent, and excluded volume plays a great role in the stabilization of apo-Mb, which does not interact with the crowders., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The interior of the cell is crowded with different kinds of biological molecules with varying sizes, shapes and compositions which may affect physiological processes especially protein folding, protein conformation and protein stability. To understand the consequences of such a crowded environment, pH-induced unfolding of holo alpha-lactalbumin (holo α-LA) was studied in the presence of ethylene glycol (EG). The effect of EG on the folding and stability of holo α-LA in aqueous solution was investigated using several spectroscopic techniques. The results indicate that stabilization/destabilization of holo α-LA by EG is concentration- and pH-dependent. Low concentration of EG stabilizes the protein at pH near its pI. From the results of far-UV CD, UV-visible and ANS fluorescence, intermediate state (MG state) was characterized in the presence of high concentration of ethylene glycol. The results invoke a new mechanism for the formation of MG state identical to active component of BAMLET. MG state of holo α-LA has a direct implication to cancer therapy. MG state of α-LA in complex with specific type of lipid is a novel class of protein-based anti-cancer complexes that incorporate oleic acid and deliver it to the cancer cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Microtubule affinity regulating kinase (MARK4) is considered as a potential drug target for diabetes, cancer, and neurodegenerative diseases. Since the role of MARK4 in the phosphorylation of tau protein and subsequently Alzheimer's disease has been established, therefore, we have investigated the binding affinity and MARK4 inhibitory potential of cholic acid (CHA) using both computational and spectroscopic methods. Molecular docking suggested a strong binding of CHA to the functionally important residues of MARK4. We further performed 500 ns molecular dynamics simulation which suggested the MARK4-CHA system was quite stable throughout the simulation trajectory. CHA potential binds to the MARK4 with a binding constant (K) of 10 7  M -1 at 288 K. Further, MARK4 activity was inhibited by CHA with an IC 50  = 5.5 μM. Further insights into the thermodynamic parameters suggested that MARK4-CHA complex formation is driven by both electrostatic and van der Waals interactions. Overall study provides a rationale to use CHA in the drug development via MARK4 inhibition, towards possible therapeutic implications in neurodegenerative diseases., Competing Interests: Declaration of competing interest The authors declare that they have no competing financial interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Known crystal structures of 10 cytochromes (cyts) c from different sources led to the conclusion that natural mutations in these proteins does not affect their 3D structure, hence evolution preserved structure for function. A sequence alignment of horse cyt c with all other 284 cyts c led to two important conclusions: (i) Leu at position 94 is conserved in all 30 mammalian known sequences, and (ii) there are 14 other species which have either Val or Ile at 94th position. We asked a question: Is the avoidance of substitution by Val or Ile at position 94 in the mammalian cyts c by design or by chance? To answer this question, we introduced natural substitutes of Leu94 by Val and Ile in horse cyt c using site-directed mutagenesis. Here, from our in vitro and molecular dynamic simulation studies on L94V and L94I mutants, we concluded that (i) although the natural mutations destabilize the wild type cyt c, it does not significantly affect the mechanism of folding of the protein, (ii) urea-induced denaturation of WT cyt c and its mutants is a two-state process, and (iii) denaturation of WT cyt c and its mutants by guanidinium chloride is not a two-state process., Competing Interests: Declaration of competing interest Authors declare there are no any financial or commercial conflict of interests with the current article content., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Do kidney osmolytes counteract the deleterious effects of urea on kidney proteins? To answer this question, we measured guanidinium chloride (GdmCl)-induced denaturation of triose phosphate isomerase-β-globin subunit complex (TIM-β-globin) from sheep kidney in the presence of various concentrations of urea and each of kidney osmolytes (glycine betaine, myo-inositol and sorbitol) alone and in combination at pH 7.5 and 25 °C. Analysis of GdmCl-induced denaturation curve at a given osmolyte (or urea) concentration gave ΔG D 0 (Gibbs free energy change in the absence of GdmCl). For a co-solute (CO), a linear plot of ΔΔG D 0 (difference between values of ΔG D 0 in the presence and absence of CO) against [CO] (molar concentration of CO) yielded m-value (=δΔΔG D 0 /δ[CO]). m-value for urea (m urea ) and that for the osmolyte (m osm ) were used to determine [urea]:[osmolyte], the molar ratio for the perfect counteraction of the denaturing effect of urea on the protein stability by an osmolyte. For each osmolyte, we tested this ratio for the perfect counteraction. It was observed that glycine betaine and myo-inositol provide perfect counteraction at the predicted molar ratio whereas sorbitol fails to do so. Furthermore, urea and GdmCl induced a two-state denaturation, and both TIM and β-globin have identical thermodynamic stability., Competing Interests: Declaration of Competing Interest Authors declare no conflict of interest., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Dextran 70 and its building block (glucose) has been used as macromolecular crowder and osmolyte, respectively. The difference in size and structure of both made us inquisitive to measure stability of lysozyme in the presence of their mixture. The effects of mixture of a fixed dextran 70 concentration (300 mg/ml) containing different concentrations of glucose and vice versa, were studied. It was observed that T m (the midpoint of denaturation curve) and △G D o (standard Gibbs free energy change at 25 °C) of lysozyme increase with the increasing concentration of dextran 70 and glucose alone and their mixture. We asked a question whether the effect of synthetic crowding agent on the stability of protein is due to the property of its monomer or due to the crowder. In this study, we observed that both dextran and its monomer stabilize the protein by same mechanism which is volume exclusion. The stabilization arises due to change in in the entropy of unfolding, while there is insignificant change in enthalpy of the protein with increasing concentration of the co-solute. Furthermore, the efficacy of stabilization by glucose increases in the crowded environment, when the concentration of dextran 70 is more than 200 mg/ml in the mixture., (Copyright © 2019 Elsevier B.V. All rights reserved.)

To understand protein folding problem under physiological condition, usually taken as dilute aqueous buffer at pH 7.0 and 25 °C, knowledge of properties of folding intermediates is important, such as molten globule (MG). We observed that polyethylene glycol 400 Da (PEG 400) induces molten globule state conformation in cytochrome c at pH 7.0 and 25 °C. This PEG-induced MG state has: (i) native tertiary structure partially perturbed, (ii) unperturbed native secondary structure, (iii) newly exposed hydrophobic patches, and (iv) has 1.58 times more hydrodynamic volume than that of the native protein. Isothermal titration calorimetry and docking studies showed specific binding between PEG 400 and cytochrome c. The study delineates that PEG-protein interactions are more complex than the excluded-volume. The soft interactions need to be seriously studied in crowding milieu that leads to destabilization of protein and overcome stabilizing exclusion volume effect. This study not only can help in unraveling the mystery of steps involved in the proper folding of proteins to solve the massively complicated problems of protein folding but also provides novel insights towards importance of structural change in proteins inside cell where intermediate states of protein import-export easily via membranes rather than native form of proteins., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020. Published by Elsevier B.V.)

The present study was aimed at investigating the binding between an important drug of Alzheimer's therapy, Rivastigmine tartrate (RT), with Bovine serum albumin (BSA). BSA is a model protein that is increasingly being used for studies related to drug-protein interaction owing to its structural similarity with human serum albumin (HSA) which is extremely abundant in the circulatory system comprising around 60% of the total plasma protein. Fluorescence spectroscopy implied that complex formation is taking place between BSA and RT; binding constant calculated was of the order of 10 4  M - 1 implicative of the strength of this interaction. Fluorescence spectroscopy was carried out at three different temperatures in a bid to find out the operative mode of quenching; static quenching was taking place for RT-BSA interaction with a binding constant of 2.5 × 10 4 M - 1 at 298 K. Further, changes in Far UV CD spectra clearly implied that RT induces structural transition in BSA suggestive of RT-BSA complex formation. The negative value of ∆G 0 as obtained from fluorescence spectroscopy and isothermal titration calorimetry (ITC) suggests the reaction to be spontaneous and thermodynamically favorable. Additionally, molecular docking was employed to investigate different forces and critical residues involved in RT-BSA interaction. Furthermore, all-atom molecular dynamics simulation for 50 ns was performed on the BSA-RT complex to investigate its conformational behavior, stability and dynamics., Competing Interests: Declaration of competing interest The authors declare that they have no competing financial interests., (Copyright © 2020. Published by Elsevier B.V.)

Sphingosine kinase 1 (SphK1) is a lipid kinase which plays vital role in the regulation of varieties of biological processes including, cell growth, apoptosis and mitogenesis. In the present study, we investigated the guanidinium chloride (GdmCl)-induced denaturation of SphK1 at pH 8.0 and 25 °C using two different spectroscopic probes, i.e., mean residue ellipticity at 222 nm ([θ] 222 ) and fluorescence emission maxima (λ max ). A significant overlap between the transition curves obtained from both the spectral properties indicate that GdmCl-induced unfolding of SphK1 follows two-state process i.e., Native (N) ⇌ Denatured (D) state. Interestingly, a visible protein aggregation was observed at low concentrations of GdmCl ([GdmCl] ≤ 1.5 M). The analysis of transition curves was done to estimate the thermodynamic parameters associated with the stability of SphK1. To complement our experimental findings, 100 ns molecular dynamics (MD) simulations were performed. Spectroscopic studies together with MD simulations provided mechanistic insights of unfolding pathway of SphK1 along with its stability parameters., Competing Interests: Declaration of competing interest Author declares no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Pyruvate dehydrogenase kinase-3 (PDK3) plays important role in the glucose metabolism and is associated with cancer progression, and thus being considered as an attractive target for cancer therapy. In this study, we employed spectroscopic techniques to study the structural and conformational changes in the PDK3 at varying pH conditions ranging from pH 2.0 to 12.0. UV/Vis, fluorescence and circular dichroism spectroscopic measurements revealed that PDK3 maintains its native-like structure (both secondary and tertiary) in the alkaline conditions (pH 7.0-12.0). However, a significant loss in the structure was observed under acidic conditions (pH 2.0-6.0). The propensity of aggregate formation at pH 4.0 was estimated by thioflavin T fluorescence measurements. To further complement structural data, kinase activity assay was performed, and maximum activity of PDK3 was observed at pH 7.0-8.0 range; whereas, its activity was lost under acidic pH. To further see conformational changes at atomistic level we have performed all-atom molecular dynamics at different pH conditions for 150 ns. A well defined correlation was observed between experimental and computational studies. This work highlights the significance of structural dependence of pH for wide implications in protein-protein interaction, biological function and drug design procedures., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Pyruvate dehydrogenase kinase 3 (PDK3) is a mitochondrial protein, has recently been considered as a potential pharmacological target for varying types of cancer. Here, we report the binding mechanism of quercetin to the PDK3 by using molecular docking, simulation, fluorescence spectroscopy and isothermal titration calorimetric assays. Molecular docking along with simulation provided an in-depth analysis of protein-ligand interactions. We have observed that quercetin interacts to the important residues of active site cavity of PDK3 and shows a well-ordered conformational fitting. The stability of quercetin-PDK3 complex is maintained by several non-covalent interactions throughout the simulation. To complement in silico findings with the experiments, we have successfully expressed and purified human PDK3. Both fluorescence and isothermal titration calorimetric experiments showed excellent binding affinity of quercetin to the PDK3. Kinase inhibition assay further revealed a significant inhibitory potential of quercetin to the PDK3 with the IC 50 values in μM range. Quercetin is non-toxic to HEK293, and significantly inhibits the proliferation of cancer (HepG2 and A549) cell lines. All these observations clearly indicate that quercetin may be further evaluated as promising therapeutic molecule for PDK3 with required modifications and in vivo validation., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Oligonucleotide/oligosaccharide-binding fold (OB-fold) plays a major role in the regulation of central dogma of life via binding though DNA and RNA. The OB-fold domains are diverse in nature and present in large number of proteins with verities of molecular functions. Here, we have investigated the distribution of sequence, structure and repeats of cold shock DNA-binding proteins (CSDB), a member of OB-fold, in all three kingdoms to establish functional relationships. The CSDB is consists of 30 domains with a major contribution of S1 (>110,601 sequences), S12 (>23,760 sequences), S17 (>14,833 sequences) and S28e (>1615 sequence) domains. These domains are largely found in bacteria (70-90%). The number of S1 domain repeats in eukaryota varies from 1 to 15 and are well-correlated with the protein size. The molecular function analysis suggests that a large number of repeats in the S1 domain are involved in diverse molecular functions in bacteria and eukaryotes. In-depth structure analysis of S1, S12, S17 and S28e domain-containing proteins of the OB-fold family provides a reasonable basis to understand the relationship of size and number of repeats with the corresponding molecular functions., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In many tissues and organisms, large amount of urea gets accumulated to maintain osmotic balance. To evade the threatening impact of urea, living organisms accumulate methylamines, a class of osmolytes, in proportion of 2:1 (urea:methylamine). To understand underlying cause(s) for protein-specific counteraction behavior, thermodynamic stability (ΔG D o ) of three disulfide free proteins (myoglobin, bovine cytochrome c and barstar) in the mixture of urea and methylamine has been estimated from guanidinium chloride-(GdmCl) driven denaturation curves. Using the experimentally measured values of ΔG D o obtained in the presence of individual methylamines and urea, we predicted the molar ratio of urea and a methylamine required for perfect compensation for each of the proteins. Interestingly, for all proteins studied, a similar ratio has been observed for perfect compensation. The predicted ratio for perfect compensation in terms of thermodynamic parameters was about 2:1 M ratio of urea to methylamine. Furthermore, a partial counteraction was observed in the myoglobin and barstar. However, for bovine cytochrome c, perfect compensation was observed in both GdmCl- and heat-driven denaturations. Our observations clearly suggest that the counteraction phenomenon depends on the extent of the unfolding of the denatured states of proteins., (Copyright © 2019. Published by Elsevier B.V.)

Organic osmolytes, as major cellular compounds, cause protein stabilization in the native form. In the present study, the possible chaperone effects of the three naturally occurring osmolytes on the two-chain form of tenecteplase (tc-TNK), a recombinant, genetically engineered mutant tissue plasminogen activator, have been explored by using circular dichroism, steady-state fluorescence, UV-Visible spectroscopy, and in silico experiments. The tc-TNK is derived from the one-chain protein upon disruption of one peptide bond. Thermal denaturation experiments showed a slightly more stabilizing effect of the three co-solvents on the single-chain TNK (sc-TNK) in comparison to that on tc-TNK. Unlike single-chain tenecteplase, the two-chain form undergoes reversible denaturation which is somehow perturbed in some cases as the result of the presence of osmolytes. Very minor changes in the secondary structure and the tertiary structure were observed. The molecular dynamics simulations and comparative structural analysis of catalytic domain of the protein in the single-chain and two-chain forms in pure water, mannitol/water, trehalose/water, and sucrose/water showed that while the stabilizing effect of the three osmolytes on tc-TNK might be induced by preferential accumulation of these molecules around the nonpolar and aromatic residues, that is to say, fewer water-hydrophobic residues' interactions in tc-TNK, sc-TNK is stabilized by preferential exclusion effect., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is an obligate pathogen that causes 10.4 million new infections worldwide, out of which about 1.4 million die every year. SDS is routinely used to mimic the native hydrophobic environment of phospholipid bilayer. Here, we report structure and stability of a mammalian cell entry protein from M. tuberculosis (Mce4A) in the absence and presence of SDS. The far-UV circular dichroism (CD) measurements suggested that SDS induces α-helical structure in Mce4A. Stability of the protein in the absence and presence of SDS was measured from the analysis of the urea-induced denaturation curves of three physical properties (CD, intrinsic fluorescence and near-UV absorption). These measurements led to the conclusion that SDS stabilizes Mce4A. Binding of SDS with Mce4A was measured in isothermal titration calorimeter, which led to the conclusion that there is strong binding of SDS with Mce4A. We propose that the membrane associated Mce4A is more structured and more stable., (Copyright © 2018. Published by Elsevier B.V.)

Myoglobin is known to show formation of intermediate states under various environmental conditions, in spite of that, this is the first evidence of formation pre-molten globule (PMG) in myoglobin. Polyethylene glycol (PEG) of various molecular sizes shows assorted effects on different proteins. Out of too short and too long PEGs, only PEGs of optimal size interact with proteins leading to change in protein structure that form intermediate state. We are the first one to report the formation of PMG in a protein in the presence of a crowding agent. The PEG-induced intermediate state was characterized by various techniques like absorption, fluorescence, near- and far-UV circular dichroism spectroscopy, ANS binding, and dynamic light scattering measurements to be PMG. Isothermal titration calorimetry and docking studies were further carried out to delineate the mechanism of formation of PMG in myoglobin in physiological conditions. The intermediate formed due to interaction of PEG with myoglobin has physiological implications which are essential to unravel the mystery to solve the massively complicated problems involved in the proper folding of proteins in vivo. Further, outcomes from this study are expected to gain mechanistic insights on the native structure and functions of proteins under in vivo conditions., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Calmodulin binding receptor like cytoplasmic kinase 2 (CRCK2) belongs to the family of receptor like kinases (RLKs) which is mainly implicated in pathways associated with the stress responses in plants. The protein from the stem of Oroxylum indicum was isolated and purified using anion-exchange followed by gel filtration chromatography. The purity of protein was checked using SDS-PAGE, which showed a single band of 50 kDa. The purified protein was identified as CRCK2 using MALDI-TOF. Using I-TASSER, a bioinformatics tools, the model of protein was constructed and its secondary structure was predicted using VADAR. The secondary structure content was also determined by far-UV CD, which indicated that the CRCK2 is mainly β-sheet dominating protein (43% β-sheet). The secondary structural content predication from computational method is in close agreement with the result obtained by CD spectropolarimeter. This study validates I-TASSER model for determination of structure of a protein. Moreover, stability of CRCK2 was monitored against heat- and guanidinium chloride (GdmCl)-induced denaturation by using circular dichroism (CD) and fluorescence spectroscopy. Denaturation curve analysis gave values of 2.88 ± 0.12 kcal mol -1 and 4.11 ± 0.09 M for ∆°G D (Gibbs free energy change at 25 °C) and C m (midpoint of denaturation), respectively. It has been observed that purified CRCK2 is quite stable protein against both heat-induced as well as GdmCl-induced denaturation. This is very first report of purification and biophysical characterization of CRCK2 protein from medicinal plant O. indicum., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Urea- and guanidinium chloride (GdmCl)-induced denatured states of horse cytochrome-c (cyt-c) are structurally identical. It is then expected that estimates of ∆G 0 N→U (Gibbs free energy change in the absence of denaturants) from GdmCl- and urea-induced denaturation curves should be identical, if denaturation induced by them follows a two-state mechanism. That denaturation of cyt-c by urea or GdmCl follows a two-state mechanism is reported by some in vitro studies while other in vitro studies reported contradictory observations. Molecular dynamic (MD) simulation is a technique that could reveal the mechanism of unfolding/folding of proteins in the absence and presence of chemical denaturants at the amino acid residue level. We therefore performed multiple unconstrained MD simulations of cyt-c (PDB ID: 1HRC) in water and aqueous mixtures of GdmCl and urea for the period of 0-500 ns at 300, 400 and 450 K, which showed that denaturation of cyt-c by urea and GdmCl is a two-state and three-state process, respectively. To corroborate these findings, we measured urea- and GdmCl-induced denaturation curves of different optical properties (circular dichroism at 222, 405 and 416 nm and absorbance at 405 nm) and analyzed them for ∆G 0 N→U . These studies supported conclusions reached from MD simulation studies., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Integrin-linked kinase (ILK) is a ubiquitously expressed Ser/Thr kinase which plays significant role in the cell-matrix interactions and growth factor signalling. In this study, guanidinium chloride (GdmCl)-induced unfolding of kinase domain of ILK (ILK 193-446 ) was carried out at pH 7.5 and 25 °C. Eventually, denaturation curves of mean residue ellipticity at 222 nm ([θ] 222 ) and fluorescence emission spectrum were analysed to estimate stability parameters. The optical properties maximum emission (λ max ) and difference absorption coefficient at 292 nm (Δε 292 ) were analysed. The denaturation curve was measured only in the GdmCl molar concentration ranging 3.0-4.2 M because protein was aggregating below 3.0 M of GdmCl concentrations. The denaturation process of ILK 193-446 was found as reversible at [GdmCl] ≥ 3.0 M. Moreover, a coincidence of normalized denaturation curves of optical properties ([θ] 222 , Δε 292 and λ max ) suggesting that GdmCl-induced denaturation of ILK 193-446 is a two-state process. In addition, 100 ns molecular dynamics simulations were performed to see the effects of GdmCl on the structure and stability of ILK 193-446 . Both the spectroscopic and molecular dynamics approaches provided clear insights into the stability and conformational properties of ILK 193-446., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Organisms synthesize sugar osmolytes during environmental stresses to protect proteins against denaturation. These studies were carried out in dilute buffer whereas intracellular milieu within cells has cytoplasmic concentration of macromolecules in the range of 80-400 mg ml -1 . Is the stabilizing effect of sugar osmolytes on the protein in dilute buffer different from that when protein is in cellular environment? To answer this question, we have measured and analysed the effect of sugar osmolytes on the structural and thermodynamic stability of ribonuclease A in the presence of dextran 70 at multiple concentrations of six sugars at different pH values. It was found that (i) each sugar osmolyte in the crowded environment provides stability to the protein in terms of T m (midpoint of denaturation) and ∆G D ° (Gibbs energy change) and this stabilizing effect is under entropic control, (ii) the extent of osmolyte-induced stabilization of RNase A is pH dependent, and (iii) effect of sugars on the stability of protein in presence of the crowding agent remains unchanged. This study concludes that crowding does not affect the efficacy of osmolytes and vice versa; and emphasizes on understanding of internal architecture of the cellular environment with respect to molecular and macromolecular crowding., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Integrin-linked kinase (ILK), a ubiquitously expressed intracellular Ser/Thr protein kinase, plays a major role in the oncogenesis and tumour progression. The conformational stability and unfolding of kinase domain of ILK (ILK 193-446 ) was examined in the presence of increasing concentrations of urea. The stability parameters of the urea-induced denaturation were measured by monitoring changes in [θ] 222 (mean residue ellipticity at 222nm), difference absorption coefficient at 292nm (Δε 292 ) and intrinsic fluorescence emission intensity at pH7.5 and 25±0.1°C. The urea-induced denaturation was found to be reversible. The protein unfolding transition occurred in the urea concentration range 3.0-7.0M. A coincidence of normalized denaturation curves of optical properties ([θ] 222 , Δε 292 and λ max , the wavelength of maximum emission intensity) suggested that urea-induced denaturation of kinase domain of ILK is a two-state process. We further performed molecular dynamics simulation for 100ns to see the effect of urea on structural stability of kinase domain of ILK at atomic level. Structural changes with increasing concentrations of urea were analysed, and we observed a significant increase in the root mean square deviation, root mean square fluctuations, solvent accessible surface area and radius of gyration. A correlation was observed between in vitro and in silico studies., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Mammalian cell entry protein (Mce4A) is a member of MCE-family, and is being considered as a potential drug target of Mycobacterium tuberculosis infection because it is required for invasion and latent survival of pathogen by utilizing host's cholesterol. In the present study, we performed molecular docking followed by 100 ns MD simulation studies to understand the mechanism of interaction of Mce4A to the cholesterol derivatives and probucol. The selected ligands, cholesterol, 25-hydroxycholesterol, 5-cholesten-3β-ol-7-one and probucol bind to the predicted active site cavity of Mce4A, and complexes remain stable during entire simulation of 100 ns. In silico studies were further validated by fluorescence-binding studies to calculate actual binding affinity and number of binding site(s). The non-toxicity of all ligands was confirmed on human monocytic cell (THP1) by MTT assay. This work provides a deeper insight into the mechanism of interaction of Mce4A to cholesterol derivatives, which may be further exploited to design potential and specific inhibitors to ameliorate the Mycobacterium pathogenesis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Alzheimer's disease (AD) is a progressive neurodegenerative disorder and the most common type of dementia in elderly ( >65years of age). Excessive extra cellular deposits of amyloid beta (Aβ) are a pathological feature of AD. Aβ can cause cell death through oxidative damage; recent studies have implicated opening of mPTP as a detrimental event in AD-related mitochondrial dysfunctions. Over the past few years, natural compounds with antioxidant properties have shown promise for intervention in AD., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Owing to the urine concentrating mechanism of kidney cells, urea concentration is very high (3.0-5.0M) in mammalian kidneys which may denature many kidney proteins. Methylamines are known to counteract the deleterious effects of urea on structure, stability and function of proteins at 2:1 molar ratio of urea to methylamines. It is known that mammalian kidney cells also contain stabilizing osmolytes, non-methylamines (myo-inositol and sorbitol). A question arises: Do these non-methylmine osmolytes have ability to counteract the deleterious effects of urea on kidney proteins? To answer this question, we took two kidney proteins, namely, sheep serum albumin and Human carbonic anhydrase II. We measured their thermodynamic stability (ΔG 0 N↔D , the Gibbs free energy change in absence of GdmCl (guanidinium chloride) associated with the equilibrium, native (N) state↔denatured (D) state) from the GdmCl-induced denaturation curves in the presence of different concentrations of urea and each kidney osmolyte individually and in combination. For both proteins, we observed that (i) glycine betaine and myo-inositol provide perfect counteraction at 2:1 molar ratio of urea to osmolyte, i.e., denaturing effect of 2M urea is 100% neutralized by 1M of glycine betaine (or myo-inositol), and (ii) sorbitol fails to refold urea denatured proteins., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Microtubule affinity regulating kinase 4 (MARK4) is a member of AMP-activated protein kinase, found to be involved in apoptosis, inflammation and many other regulatory pathways. Since, its aberrant expression is directly associated with the cell cycle and thus cancer. Therefore, MARK4 is being considered as a potential drug target for cancer therapy. Here, we investigated the mechanism of inhibition of MARK4 activity by citral. Docking studies suggested that citral effectively binds to the active site cavity, and complex is stabilized by several interactions. We further performed molecular dynamics simulation of MARK4-citral complex under explicit water condition for 100ns and observed that binding of citral to MARK4 was quite stable. Fluorescence binding studies suggested that citral strongly binds to MARK4 and thereby inhibits its enzyme activity which was measured by the kinase inhibition assay. We further performed MTT assay and observed that citral inhibits proliferation of breast cancer cell line MCF-7. This work provides a newer insight into the use of citral as novel cancer therapeutics through the MARK4 inhibition. Results may be employed to design novel therapeutic molecule using citral as a scaffold for MARK4 inhibition to fight related diseases., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Here, we report the formation of molten globule state of the native myoglobin in crowded environment. We have used Soret absorption spectroscopy and far-UV circular dichroism to monitor changes in tertiary and secondary structures of myoglobin, respectively. Our results reveal that in the presence of ficoll 70, the secondary structure of myoglobin remains unchanged while tertiary structure is lost significantly. 1-anilinonaphthalene-8-sulfonate binding experiments showed that myoglobin in the presence of various concentrations of ficoll 70, has newly exposed hydrophobic surfaces. Dynamic light scattering measurements show that there is almost 1.5 times increase in the hydrodynamic volume of myoglobin in the crowded environment. These structural characteristics of myoglobin in the presence of 300mg/ml ficoll 70 resemble those of molten globule state. Isothermal titration calorimetric (ITC) measurements show that ficoll 70 binds to myoglobin, whereas it shows no interaction with apo form of the protein. ITC results indicate that the reason behind this unique behavior of ficoll 70 towards myoglobin may be interaction of ficoll 70 with the heme group of myoglobin, which was further confirmed by the docking studies. We hypothesize that the soft interactions between heme and ficoll 70 leads to the formation of molten globule in myoglobin., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Carbonic anhydrase IX (CAIX) is a transmembrane glycoprotein, overexpressed in cancer cells under hypoxia condition. In cancerous cells, CAIX plays an important role to combat the deleterious effects of a high rate of glycolytic metabolism. In order to favor tumor survival, CAIX maintains intracellular pH neutral or slightly alkaline and extracellular acidic pH. The equilibrium unfolding and conformational stability of CAIX were measured in the presence of increasing urea concentrations to understand it's structural features under stressed conditions. Two different spectroscopic techniques were used to follow urea-induced denaturation and observed that urea induces a reversible denaturation of CAIX. Coincidence of the normalized transition curves of both optical properties suggesting that denaturation of CAIX is a two-state process, i.e., native state ↔ denatured state. Each denaturation curve was analyzed to estimate thermodynamic parameters, ΔG D 0 ,value of Gibbs free energy change (ΔG D ) associated with the urea-induced denaturation, C m (midpoint of denaturation) and m (=δΔG D /δ[urea]). We further performed molecular dynamics simulation of CAIX for 50ns to see the dynamics of protein structure in the presence of different urea concentrations. An excellent agreement was observed between in silico and in vitro studies., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The macromolecular crowding progressively has been gaining prominence in recent years as it acts as a sword with double-edge on protein stability and folding, i.e., showing assorted results of having both stabilizing and destabilizing effects. We studied the effects of different concentrations of polyethylene glycol (PEG-10) on structure and stability of myoglobin. The tertiary structure of myoglobin was found to be perturbed in the presence of polyethylene glycol, however there was insignificant change in the secondary structure. It was observed that polyethylene glycol induces molten globule state in myoglobin, where the intermediate state holds hydrophobic patches and larger hydrodynamic volume as compared to the native protein. In addition, isothermal titration calorimetry (ITC) showed strong binding between myoglobin and polyethylene glycol, at the physiological pH. We hypothesize that polyethylene glycol induces molten globule conformation in myoglobin by interacting with heme group of myoglobin. We caution that the binding of protein with crowder and other soft interactions need to be gravely well thought-out when studying macromolecular crowding. In our case, destabilizing protein-crowder interactions could compete and overcome the stabilizing exclusion volume effect., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Recently, we had published that urea-induced denaturation curves of optical properties of sheep serum albumin (SSA) are biphasic with a stable intermediate that has characteristics of molten globule (MG) state. In this study, we have extended the work by carrying out urea- and guanidinium chloride (GdmCl)-induced denaturations of SSA in the presence of naturally occurring mammalian kidney osmolytes, namely, sorbitol, myo-inositol and glycine betaine. We have observed that all these osmolytes (i) transform this biphasic transition into a co-operative, two-state transition and (ii) increase the stability of the protein in terms of midpoint of denaturation (C m ) and Gibbs free energy change in the absence of both denaturants (ΔG D 0 ). The relative effectiveness of different osmolytes on the stability of SSA follows the order: glycine betaine>myo-inositol>sorbitol. In this paper, we also report that kidney osmolytes destabilize MG state by shifting the equilibrium, native state↔MG state toward the left. This study will be helpful in understanding the existence of osmolytes in kidney and their role in folding of kidney proteins soaked with urea., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Human calcium/calmodulin-dependent protein kinase IV (CAMKIV), a member of Ser/Thr kinase family, is associated with cancer, cerebral hypoxia and neurodegenerative diseases. β-carotene is a colored organic compound, abundant in plants and fruits and is used in cancer prevention. Here, we report a strong binding affinity of β-carotene with CAMKIV using molecular docking, fluorescence binding and isothermal titration calorimetry methods. Furthermore, β-carotene also reduces the enzyme activity of CAMKIV moderately as observed during ATPase assay. To see the role of β-carotene on cell proliferation and apoptosis, cancerous cells (HeLa, HuH7and MCF-7) and normal (HEK-293-T) cell lines were used. Admirable anticancer activity of β-carotene was observed. We further performed propidium iodide and DAPI (4',6-diamidino-2-phenylindole) assays to understand the mechanism of anticancer activity of β-carotene at molecular level. Our findings provide a newer insight into the use of β-carotene in cancer prevention and protection via inhibition of CAMKIV by regulating the signaling pathways., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Knowledge of folding/unfolding pathway is fundamental basis to study protein structure and stability. Human carbonic anhydrase II (HCAII) is a ∼29kDa, β-sheet dominated monomeric protein of 259 amino acid residues. In the present study, the urea-induced denaturation of HCAII was carried out which was a tri-phasic process, i.e., N (native) ↔ X I ↔ X II ↔ D (denatured) with stable intermediates X I and X II populated around 2 and 4M urea, respectively. The far-UV CD was used to characterize the intermediate states (X I and X II ) for secondary structural content, near-UV CD for tertiary structure, dynamic light scattering for hydrodynamic radius and ANS fluorescence spectroscopy for the presence of exposed hydrophobic patches. Based on these experiments, we concluded that urea-induced X I state has characteristics of molten globule state while X II state bears characteristics features of pre-molten globule state. Characterization of the intermediates on the folding pathway will contribute to a deeper understanding of the structure-function relationship of HCAII. Furthermore, this system may provide an excellent model to study urea stress and the strategies adopted by the organisms to combat such a stress., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Triose phosphate isomerase (TIM) is a cytoplasmic enzyme of prime importance in the mammalian glycolytic pathway. It has a major role in the conversion of dihydroxyacetone phosphate into glyceraldehyde-3-phosphate. We have successfully purified a stable complex of TIM with β-globin subunit from the sheep kidney using a simple two-step chromatography procedure. It is seen for the first time that TIM is forming a stable complex with β-globin. The purified protein-protein complex was crystallized and preliminary diffraction data were collected at 2.1Å resolution. We further studied guanidinium chloride (GdmCl)-induced denaturation of TIM-β-globin complex by monitoring changes in the mean residue ellipticity at 222nm ([θ] 222 ) and difference absorption coefficient at 406nm (Δε 406 ) at pH 7.5 and 25°C. We have observed that GdmCl-induced denaturation is reversible. Coincidence of normalized transition curves of both physical properties ([θ] 222 and Δε 406 ) suggests that folding/unfolding of TIM and β-subunit proteins is a two-state process. Denaturation curves of [θ] 222 and Δε 406 were used to estimate the stability parameters of the protein-protein complex. This is the first report on the isolation, purification, crystallization and biophysical characterization of the naturally occurring complex of TIM with the β-globin subunit., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The mce4A gene of Mycobacterium tuberculosis encodes a 400 amino acid residues protein of 43kDa, which is a mammalian cell entry protein (Mce4A) and plays important role in host cell invasion. Mce4A helps in long-term survival of M. tuberculosis by cholesterol utilization. Host cholesterol utilization mechanism by Mce4A is not clearly understood. In order to investigate the role of Mce4A in M. tuberculosis pathogenesis, we purified the recombinant protein by affinity chromatography, analyzed by SDS-PAGE and confirmed by western blot. We performed structural studies of Mce4A as function of pH and salt concentration by using different spectroscopic techniques. This protein was found to be stable over the wide range of pH 5.5≤pH≤11.5. An addition of sodium chloride up to the concentration of 150mM, shows no significant change in the secondary structure content of the protein. To confirm its activity, we performed isothermal titration calorimetry measurements of Mce4A in the presence of cholesterol. This is the first report of binding of cholesterol to Mce4A in vitro. Binding of cholesterol to Mce4A is sequential four-step and entropy driven process. The structural studies of this protein will help to understand the mechanism of pathogenesis of M. tuberculosis., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Microtubule affinity regulating Kinase 4 (MARK4) belongs to the family of AMP-activated protein kinase. It phosphorylates microtubule associated proteins at specific sites (Serine in KXGS motifs) in the microtubule-binding repeats. In our previous studies, two constructs, namely, kinase domain with 59 N-terminal residues (residues 1-310) and only kinase domain (residues 59-310) of MARK4 show aggregation at physiological pH. However, these two constructs were stable at extremes of pH conditions. Now the question arises: how is MARK4 stable at physiological pH in-vivo? To answer this question, we have successfully cloned, expressed, and purified UBA-domain along with the kinase domain of MARK4 and performed spectroscopic measurements and activity assays. We observed a pronounced secondary and tertiary structure and ATPase activity in the MARK4 at physiological pH. In conclusion, UBA domain may be important to maintain the structure, stability and activity of MARK4 under physiological conditions., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Mammalian mitochondrial cytochromes c (cyts c) has a conserved Leu94 which is replaced by valine/isoleucine in some lower eukaryotes and prokaryotes. It is expected that nature substituted Leu94 with Val/Ile, for they have similar van der Waals volume and hydrophobicity with a difference in side chain branching only. Reports also suggested the presence of phenylalanine at position 94, which leads to questions: (i) How bulky aromatic amino acid residue fitted at position 94 in cyt c family proteins? (ii) What is the effect of L94F mutation on protein stability and folding? Here, we selected horse cyt-c as a model to answer the second question. We generated L94F mutant of horse cytochrome c and subsequently characterised using far-UV, near-UV and Soret circular dichroism, absorbance, intrinsic and extrinsic ANS (8-anilino-1-napthalenesulfonic acid) fluorescence and dynamic light scattering measurements. We observed that this mutation affects the native state and arrests the protein folding at the molten globule state. Thermal stability of L94F mutant is also measured by spectroscopic techniques and differential scanning calorimetry. The midpoint of thermal denaturation of L94F mutant is 17°C less than wild type. Molecular dynamics simulation study also supports our in vitro observation that this mutant has stable backbone conformation., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Carbonic anhydrase VA (CAVA) is a mitochondrial enzyme belonging to the α-family of CAs, which is involved in several physiological processes including ureagenesis, lipogenesis, gluconeogenesis and neuronal transmission. Here, we have tried to understand the folding mechanism of CAVA using guanidine hydrochloride (GdnHCl)-induced denaturation at pH 8.0 and 25°C. The conformational stability was measured from the GdnHCl-induced denaturation study of CAVA monitored by circular dichroism (CD) and fluorescence measurements. On increasing the concentration of GdnHCl up to 5.0, a stable intermediate was observed between the concentrations 3.25M to 3.40M of the denaturant. However, CAVA gets completely denatured at 4.0M GdnHCl. The existence of a stable intermediate state was validated by 1-anilinonaphthalene-8-sulfonic acid (ANS binding) fluorescence and near-UV CD measurements. In silico studies were also performed to analyse the effect of GdnHCl on the structure and stability of CAVA under explicit conditions. Molecular dynamics simulations for 40ns were carried out and a well-defined correlation was established for both in vitro and in silico studies., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Hereditary hemochromatosis factor E (HFE) is a type 1 transmembrane protein, and acts as a negative regulator of iron-uptake. The equilibrium unfolding and conformational stability of the HFE protein was examined in the presence of urea. The folding and unfolding transitions were monitored with the help of circular dichroism (CD), intrinsic fluorescence and absorption spectroscopy. Analysis of transition curves revealed that the folding of HFE is not a two-state process. However, it involved stable intermediates. Transition curves (plot of fluorescence (F346) and CD signal at 222nm (θ222) versus [Urea], the molar urea concentration) revealed a biphasic transition with midpoint (Cm) values at 2.88M and 4.95M urea. Whereas, absorption analysis shows one two-state transition centered at 2.96M. To estimate the protein stability, denaturation curves were analyzed for Gibbs free energy change in the absence of urea (ΔGD(0)) associated with the equilibrium of denaturation exist between native state↔denatured state. The intermediate state was further characterized by hydrophobic probe, 1-anilinonaphthalene-8-sulfonic acid (ANS-binding). For seeing the effect of urea on the structure and dynamics of HFE, molecular dynamics simulation for 60ns was also performed. A clear correspondence was established between the in vitro and in silico studies., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Sheep serum albumin (SSA) is a 583 amino acid residues long multidomain monomeric protein which is rich in cysteine and low in tryptophan content. The serum albumins (from human, bovine and sheep) play a vital role among all proteins investigated until now, as they are the most copious circulatory proteins. We have purified SSA from sheep kidneys by a simple and efficient two-step purification procedure. Further, we have studied urea-induced denaturation of SSA by monitoring changes in the difference absorption coefficient at 287nm (Δε287), intrinsic fluorescence emission intensity at 347nm (F347) and mean residue ellipticity at 222nm ([θ]222) at pH 7.4 and 25°C. The coincidence of denaturation curves of these optical properties suggests that urea-induced denaturation is a bi-phasic process (native (N) state↔intermediate (X) state↔denatured (D) state) with a stable intermediate populated around 4.2-4.7M urea. The intermediate (X) state was further characterized by the far-UV and near-UV CD, dynamic light scattering (DLS) and fluorescence using 1-anilinonaphthalene-8-sulfonic acid (ANS) binding method. All denaturation curves were analyzed for Gibbs free energy changes associated with the equilibria, N state↔X state and X state↔D state in the absence of urea., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Cyanobacterial phycoerythrin (αC-PE) from Phormidium tenue exists in two natural forms named as full length (FL-αC-PE) and truncated (Tr-αC-PE). FL-αC-PE and Tr-αC-PE are produced when cyanobacterium is grown in the optimal medium and nutrient deficient medium, respectively. Despite of N-terminal deletion, both proteins show similar spectroscopic properties. In this study, different optical properties of these two natural variants of C-PE were measured in the pH range 1.0-12.0 (1.0 ≤ pH ≤ 12.0). It was observed that: (i) their absorption, fluorescence and CD spectra remain unchanged within the range adjacent to neutral pH, 5.5-8.75, (ii) at pH values higher than 8.75 and lower than 5.5 their absorption, fluorescence and CD spectral signatures are changed significantly, and (iii) emission spectra of the covalently linked tetrapyrrole chromophores and Trp residue are perturbed at extreme pH values in the range 8.75