Beyond Glucose: The Dual Assault of Oxidative and ER Stress in Diabetic Disorders.

Diabetes mellitus, a prevalent global health concern, is characterized by hyperglycemia. However, recent research reveals a more intricate landscape where oxidative stress and endoplasmic reticulum (ER) stress orchestrate a dual assault, profoundly impacting diabetic disorders. This review elucidates the interplay between these two stress pathways and their collective consequences on diabetes. Oxidative stress emanates from mitochondria, where reactive oxygen species (ROS) production spirals out of control, leading to cellular damage. We explore ROS-mediated signaling pathways, which trigger ß-cell dysfunction, insulin resistance, and endothelial dysfunction the quintessential features of diabetes. Simultaneously, ER stress unravels, unveiling how protein folding disturbances activate the unfolded protein response (UPR). We dissect the UPR's dual role, oscillating between cellular adaptation and apoptosis, significantly influencing pancreatic ß-cells and peripheral insulin-sensitive tissues. Crucially, this review exposes the synergy between oxidative and ER stress pathways. ROS-induced UPR activation and ER stress-induced oxidative stress create a detrimental feedback loop, exacerbating diabetic complications. Moreover, we spotlight promising therapeutic strategies that target both stress pathways. Antioxidants, molecular chaperones, and novel pharmacological agents offer potential avenues for diabetes management. As the global diabetes burden escalates, comprehending the dual assault of oxidative and ER stress is paramount. This review not only unveils the intricate molecular mechanisms governing diabetic pathophysiology but also advocates a holistic therapeutic approach. By addressing both stress pathways concurrently, we may forge innovative solutions for diabetic disorders, ultimately alleviating the burden of this pervasive health issue.

Potential antiviral activities of chrysin against hepatitis B virus.

BACKGROUND: Interferon and nucleos(t)ide analogues are current therapeutic treatments for chronic Hepatitis B virus (HBV) infection with the limitations of a functional cure. Chrysin (5, 7-dihydroxyflavone) is a natural flavonoid, known for its antiviral and hepatoprotective activities. However, its anti-HBV activity is unexplored. METHODS: In the present study, the anti-hepatitis B activity of chrysin was investigated using the in vitro experimental cell culture model, HepG2 cells. In silico studies were performed where chrysin and lamivudine (used here as a positive control) were docked with high mobility group box 1 protein (HMGB1). For the in vitro studies, wild type HBV genome construct (pHBV 1.3X) was transiently transfected in HepG2. In culture supernatant samples, HBV surface antigen (HBsAg) and Hepatitis B e antigen (HBeAg) were measured by enzyme-linked immunosorbent assay (ELISA). Secreted HBV DNA and intracellular covalently closed circular DNA (cccDNA) were measured by SYBR green real-time PCR. The 3D crystal structure of HMGB1 (1AAB) protein was developed and docked with the chrysin and lamivudine. In silico drug-likeness, Absorption, Distribution, Metabolism, Excretion and Toxicity (ADMET) properties of finest ligands were performed by using SwissADME and admetSAR web servers. RESULTS: Data showed that chrysin significantly decreases HBeAg, HBsAg secretion, supernatant HBV DNA and cccDNA, in a dose dependent manner. The docking studies demonstrated HMGB1 as an important target for chrysin as compared to lamivudine. Chrysin revealed high binding affinity and formed a firm kissing complex with HMGB1 (∆G = - 5.7 kcal/mol), as compared to lamivudine (∆G = - 4.3 kcal/mol), which might be responsible for its antiviral activity. CONCLUSIONS: The outcome of our study establishes chrysin as a new antiviral against HBV infection. However, using chrysin to treat chronic HBV disease needs further endorsement and optimization by in vivo studies in animal models.

Elucidating the Role of Santalol as a Potent Inhibitor of Tyrosinase: In Vitro and In Silico Approaches.

This research work focuses on the potential application of an organic compound, santalol, obtained from santalum album, in the inhibition of the enzyme tyrosinase, which is actively involved in the biosynthesis of melanin pigment. Over-production of melanin causes undesirable pigmentation in humans as well as other organisms and significantly downgrades their aesthetic value. The study is designed to explain the purification of tyrosinase from the mushroom Agaricus bisporus, followed by activity assays and enzyme kinetics to give insight into the santalol-modulated tyrosinase inhibition in a dose-dependent manner. The multi-spectroscopic techniques such as UV-vis, fluorescence, and isothermal calorimetry are employed to deduce the efficiency of santalol as a potential candidate against tyrosinase enzyme activity. Experimental results are further verified by molecular docking. Santalol, derived from the essential oils of santalum album, has been widely used as a remedy for skin disorders and a potion for a fair complexion since ancient times. Based on enzyme kinetics and biophysical characterization, this is the first scientific evidence where santalol inhibits tyrosinase, and santalol may be employed in the agriculture, food, and cosmetic industries to prevent excess melanin formation or browning.

Measuring Structural Changes in Cytochrome c under Crowded Conditions Using In Vitro and In Silico Approaches.

It is known from in vitro studies that macromolecular crowding in the cell effects protein structure, stability and function; but predictive studies are relatively unexplored. There are few reports where the effect of various crowder mixtures has been exploited to discern their combined effect on the structural stability of proteins. These studies are more significant because their effect can mimicked with in vivo conditions, where the environment is heterogeneous. Effects of two crowders, polyethylene glycol (PEG 400 Da), and its monomer ethylene glycol (EG) alone and in mixture on the structural stability of cytochrome c (cyt c) were determined using various spectroscopic and bioinformatics tools. The main conclusions of our study are (i) the monomer EG has a kosmotropic effect on the protein (stabilizes the protein), and has no significant effect on the tertiary structure; (ii) PEG 400 destabilizes the structure as well as the stability of the protein; and (iii) EG counteracts the destabilizing effect of PEG 400. From this investigation, it seems evident that proteins may fold or unfold in the crowded environment of the cell where various interactions assist them to maintain their structure for their functions. Bioinformatics approaches were also used to support all of the in vitro observations. Cyt c is functional protein; if the structure of the protein is modulated due to change in the environment its nature of function will also change. Our research addresses the question by modulating the environment around the protein, and the macromolecule (protein) conformation dynamics and interaction study via in vitro and in silico approaches which indirectly compares with that of the environment in-cellular milieu, which is highly crowded.

Size-Dependent Interplay of Volume Exclusion Versus Soft Interactions: Cytochrome c in Macromolecular Crowded Environment.

Even though there are a great number of possible conformational states, how a protein generated as a linear unfolded polypeptide efficiently folds into its physiologically active form remained a fascinating and unanswered enigma inside crowded conditions of cells. In this study, various spectroscopic techniques have been exploited to know and understand the effect and mechanism of action of two different sizes of polyethylene glycols, or PEGs (molecular mass ∼10 and ∼20 kilo Daltons, kDa), on cytochrome c (cyt c). The outcomes showed that small size of the PEG leads to perturbation of the protein structure, and conversely, large size of the PEG has stabilizing effect on cyt c. Moreover, binding measurements showed that small size of PEG interacts strongly via soft interactions compared to the larger size of PEG, the latter being governed more by excluded volume effect or preferential exclusion from the protein. Overall, this finding suggests that conformations of protein may be influenced in cellular crowded conditions via interactions which depend upon the size of molecule in the environment. This study proposes that both volume exclusion and soft (chemical) interactions governs the protein's conformation and functional activities. The cellular environment's internal architecture as evident from crowder size and shape in this study has a significant role.

Insights into Fluctuations of Structure of Proteins: Significance of Intermediary States in Regulating Biological Functions.

Proteins are indispensable to cellular communication and metabolism. The structure on which cells and tissues are developed is deciphered from proteins. To perform functions, proteins fold into a three-dimensional structural design, which is specific and fundamentally determined by their characteristic sequence of amino acids. Few of them have structural versatility, allowing them to adapt their shape to the task at hand. The intermediate states appear momentarily, while protein folds from denatured (D) ⇔ native (N), which plays significant roles in cellular functions. Prolific effort needs to be taken in characterizing these intermediate species if detected during the folding process. Protein folds into its native structure through definite pathways, which involve a limited number of transitory intermediates. Intermediates may be essential in protein folding pathways and assembly in some cases, as well as misfolding and aggregation folding pathways. These intermediate states help to understand the machinery of proper folding in proteins. In this review article, we highlight the various intermediate states observed and characterized so far under in vitro conditions. Moreover, the role and significance of intermediates in regulating the biological function of cells are discussed clearly.

Mutation in the CX3C Motif of G Protein Disrupts Its Interaction with Heparan Sulfate: A Calorimetric, Spectroscopic, and Molecular Docking Study.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection in children and infants. To date, there is no effective vaccine available against RSV. Heparan sulfate is a type of glycosaminoglycan that aids in the attachment of the RSV to the host cell membrane via the G protein. In the present study, the effect of amino acid substitution on the structure and stability of the ectodomain G protein was studied. Further, it was investigated whether mutation (K117A) in the CX3C motif of G protein alters the binding with heparan sulfate. The point mutation significantly affects the conformational stability of the G protein. The mutant protein showed a low binding affinity with heparan sulfate as compared to the wild-type G protein, as determined by fluorescence quenching, isothermal titration calorimetry (ITC), and molecular docking studies. The low binding affinity and decreased stability suggested that this mutation may play an important role in prevention of attachment of virion to the host cell receptors. Collectively, this investigation suggests that mutation in the CX3C motif of G protein may likely improve the efficacy and safety of the RSV vaccine.

Structural Refolding and Thermal Stability of Myoglobin in the Presence of Mixture of Crowders: Importance of Various Interactions for Protein Stabilization in Crowded Conditions.

The intracellular environment is overcrowded with a range of molecules (small and large), all of which influence protein conformation. As a result, understanding how proteins fold and stay functional in such crowded conditions is essential. Several in vitro experiments have looked into the effects of macromolecular crowding on different proteins. However, there are hardly any reports regarding small molecular crowders used alone and in mixtures to observe their effects on the structure and stability of the proteins, which mimics of the cellular conditions. Here we investigate the effect of different mixtures of crowders, ethylene glycol (EG) and its polymer polyethylene glycol (PEG 400 Da) on the structural and thermal stability of myoglobin (Mb). Our results show that monomer (EG) has no significant effect on the structure of Mb, while the polymer disrupts its structure and decreases its stability. Conversely, the additive effect of crowders showed structural refolding of the protein to some extent. Moreover, the calorimetric binding studies of the protein showed very weak interactions with the mixture of crowders. Usually, we can assume that soft interactions induce structural perturbations while exclusion volume effects stabilize the protein structure; therefore, we hypothesize that under in vivo crowded conditions, both phenomena occur and maintain the stability and function of proteins.

Structural Characterization and Binding Studies of the Ectodomain G Protein of Respiratory Syncytial Virus Reveal the Crucial Role of pH with Possible Implications in Host-Pathogen Interactions.

Respiratory syncytial virus (RSV) is a leading viral pathogen causing acute lower respiratory tract infection in children. The G protein of RSV is involved in attachment with the host cell. It is a neutralizing antigen and thus a vaccine candidate. Heparan sulfate is a type of glycosaminoglycan (GAG) present on the host cell membrane that is involved in attachment with the G protein of RSV. We describe a novel approach for efficient expression and purification of the ectodomain G protein in the prokaryotic system and its biophysical characterization. The native ectodomain G protein was purified using a two-step process by Ni-NTA and DEAE weak anion-exchange chromatography through the supernatant obtained after cell lysis. In addition, the denatured form of the protein was also purified from the solubilized inclusion bodies (IBs) by Ni-NTA affinity chromatography with a higher yield. Dynamic light scattering (DLS) was performed to confirm the homogeneity of the purified protein. The effect of pH on the stability and structure of the purified protein was studied by circular dichroism (CD), fluorescence, and absorbance spectroscopy techniques. Isothermal titration calorimetry (ITC) and microscale thermophoresis (MST) were exploited to demonstrate the interaction of heparan sulfate with the ectodomain G protein. The dynamic light scattering results showed that the purified protein was homogenic and had a well-folded native conformation. Biophysical characterization of the protein revealed that it was stable and had intact secondary and tertiary structures at pH 7.5. CD analysis revealed that the protein showed a loss in the secondary structure at pH values 5.5 and 3.5, while absorbance spectroscopy suggested a stable tertiary structure at pH values 7.5 and 5.5 with a probable aggregation pattern at pH 3.5. This loss in the structure of the ectodomain G protein at low pH can be correlated with its physiological activity. A slight change in pH might play a crucial role in host-pathogen interactions. The fluorescence intensity of the protein decreased on moving toward a lower pH with no spectral shift in emission maxima. In addition, isothermal titration calorimetry and microscale thermophoresis results showed strong binding affinity of the ectodomain G protein with heparan sulfate. The binding of heparan sulfate with protein was probably due to the electrostatic interaction of positively charged amino acid residues of the heparin-binding domain of the protein and the negatively charged group of GAGs. Future studies may involve the development of possible therapeutic agents interacting with the G protein and affecting the overall charge and pH that might hinder the host-pathogen interaction.

Interaction of polyethylene glycol with cytochrome c investigated via in vitro and in silico approaches.

One of the significant proteins that have attracted research groups due to virtue of being a potent selective anticancer drug target and property of triggering apoptosis upon release in cytoplasm is cytochrome c (cyt c). The mechanical transformations due to the macromolecular crowding in membrane in the mammalian cell are proposed to be useful inductors of changes in volume. It is very interesting to know that mitochondrial function were observed to be improved by polyethylene glycol (PEG) interaction, which in turn inhibits the cyt c (a pro-apoptotic cell death factor). In this work, the effect of polyethylene glycol of molecular weight 4 kilo Dalton (PEG 4 kDa) was investigated to highlight the structural transformations (tertiary and secondary structure) in cyt c using a choice of spectroscopic techniques (including UV-Vis absorption, near-UV, far-UV and Soret circular dichroism and fluorescence spectroscopy), which shows noteworthy shifts in the secondary and tertiary structures at higher concentrations of PEG 4 kDa with small changes in the heme-globular interactions. The size distribution changes of native protein treated with various concentrations of the crowder were observed and analyzed by dynamic light scattering (DLS). The interaction studies of the crowder with the protein was observed and analyzed by FTIR, isothermal titration calorimetry, time resolved fluorescence and molecular docking. The investigations suggested that the structural changes in the protein occurred due to soft interactions of PEG 4 kDa, which usually destabilizes proteins. The experimental evidence in this study proposed that crowding could be another approach to mechanical super-competition and free of certain markers that could aid in the identification and control of various diseases. This study suggests that crowders at specific concentrations, which softly interact with proteins, can be exploited as remedy for various diseases.

Crowding Milleu stabilizes apo-myoglobin against chemical-induced denaturation: Dominance of hardcore repulsions in the heme devoid protein.

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 Δε291 and θ222 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 (Cm) and change in free energy in the absence of denaturant (∆GD°), 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.

Rationalizing the Role of Monosodium Glutamate in the Protein Aggregation Through Biophysical Approaches: Potential Impact on Neurodegeneration.

Monosodium glutamate (MSG) is the world's most extensively used food additive and is generally recognized as safe according to the FDA. However, it is well reported that MSG is associated with a number of neurological diseases, and in turn, neurological diseases are associated with protein aggregation. This study rationalized the role of MSG in protein aggregation using different biophysical techniques such as absorption, far-UV CD, DLS, and ITC. Kinetic measurements revealed that MSG causes significant enhancement of aggregation of BSA through a nucleation-dependent polymerization mechanism. Also, CTAB-BSA aggregation is enhanced by MSG significantly. MSG-induced BSA aggregation also exhibits the formation of irreversible aggregates, temperature dependence, non-Arrhenius behavior, and enhancement of hydrodynamic diameter. From the isothermal titration calorimetry measurement, the significant endothermic heat of the interaction of BSA-MSG indicates that protein aggregation may be due to the coupling of MSG with the protein. The determined enthalpy change (ΔH) is largely positive, also suggesting an endothermic nature, whereas entropy change (ΔS) is positive and Gibbs free energy change (ΔG) is largely negative, suggesting the spontaneous nature of the interaction. Furthermore, even a low concentration of MSG is involved in the unfolding of the secondary structure of protein with the disappearance of original peaks and the formation of a unique peak in the far-UV CD, which is an attention-grabbing observation. This is the first investigation which links the dietary MSG with protein aggregation and thus will be very instrumental in understanding the mechanism of various MSG-related human physiological as well as neurological diseases.

Heparin Accelerates the Protein Aggregation via the Downhill Polymerization Mechanism: Multi-Spectroscopic Studies to Delineate the Implications on Proteinopathies.

Heparin is one of the members of the glycosaminoglycan (GAG) family, which has been associated with protein aggregation diseases including Alzheimer's disease, Parkinson's disease, and prion diseases. Here, we investigate heparin-induced aggregation of bovine serum albumin (BSA) using different spectroscopic techniques [absorption, 8-anilino-1-naphthalene sulfonic acid (ANS) and thioflavin T (ThT) fluorescence binding, and far- and near-UV circular dichroism]. Kinetic measurements revealed that heparin is involved in the significant enhancement of aggregation of BSA. The outcomes showed dearth of the lag phase and a considerable change in rate constant, which provides conclusive evidence, that is, heparin-induced BSA aggregation involves the pathway of the downhill polymerization mechanism. Heparin also causes enhancement of fluorescence intensity of BSA significantly. Moreover, heparin was observed to form amyloids and amorphous aggregates of BSA which were confirmed by ThT and ANS fluorescence, respectively. Circular dichroism measurements exhibit a considerable change in the secondary and tertiary structure of the protein due to heparin. In addition, binding studies of heparin with BSA to know the cause of aggregation, isothermal titration calorimetry measurements were exploited, from which heparin was observed to promote the aggregation of BSA by virtue of electrostatic interactions between positively charged amino acid residues of protein and negatively charged groups of GAG. The nature of binding of heparin with BSA is very much apparent with an appreciable heat of interaction and is largely exothermic in nature. Moreover, the Gibbs free energy change (ΔG) is negative, which indicates spontaneous nature of binding, and the enthalpy change (ΔH) and entropy change (ΔS) are also largely negative, which suggest that the interaction is driven by hydrogen bonding.

Conformational changes in cytochrome c directed by ethylene glycol accompanying complex formation: Protein-solvent preferential interaction or/and kosmotropic effect.

When proteins interact with solvent or co-solutes with a high specificity and affinity, protein-ligand complexes may be formed. Such phenomenon may involve the processes like intra- and intermolecular interactions, which result in interaction based protein folding. In this study, cytochrome c (cyt c) was treated with different concentrations of ethylene glycol (EG) in crowded and confined media to check its structural stability using various spectroscopic techniques at pH 7.0 and 25 °C. The various spectroscopic techniques including circular dichroism (Soret, far- and near-UV regions), Fourier transform infrared (FTIR), absorption (UV and visible) and Trp fluorescence shows both secondary and tertiary structure of cyt c increases when treated with EG. The investigations using dynamic light scattering (DLS), time resolved fluorescence and isothermal titration calorimetry (ITC) for binding studies shows weak interaction between EG and cyt c. Small increase in the structure of the protein and insignificant decrease in hydrodynamic radii of the protein was observed from the studies. Molecular docking studies showed that EG has binding site on the protein and interact with few amino acid residues by weak interactions such as van der Waals and hydrogen bonding. This study helps in understanding the protein-ligand interactions, provides facts and the mechanisms that mediates the recognition of binding site for specific ligand to the receptor protein, which make possible of the discovery, design, and development of drugs at molecular level without affecting proteins within an organism.

Effects of Ethylene Glycol on the Structure and Stability of Myoglobin Using Spectroscopic, Interaction, and In Silico Approaches: Monomer Is Different from Those of Its Polymers.

Investigation of changes in thermal stabilities and structures of proteins in the presence of different co-solutes (ligands) is an integral part in the basic research, discovery, and development of drugs. Ethylene glycol (EG) is known to be toxic and causes teratogenic, inducing primarily skeletal and external malformations and other diseases. The effect of EG on the structure and thermal stability of myoglobin (Mb) was studied using various spectroscopic techniques at pH 7.0 and two different temperatures. As revealed by circular dichroism, Trp fluorescence, nano-DSF, and absorption (UV and visible) measurements, EG (i) has no significant effect on secondary and tertiary structures of Mb at 25 °C, and (ii) it decreases the thermal stability of the protein, which increases with increasing concentration of EG. As revealed by ANS (8-anilino-1-naphthalene sulfonic acid) fluorescence measurements, heat-induced denatured protein has newly exposed hydrophobic patches that bind to ANS. Isothermal titration calorimetry revealed that the interaction between EG and Mb is temperature dependent; the preferential interaction of EG is entropy driven at low temperature, 298 K (25 °C), and it is enthalpy driven at higher temperature, 343 K (70 °C). Molecular docking study showed that EG interacts with side chains of amino acid residues of Mb through van der Waals interactions and hydrogen bonding.

Interactions Under Crowding Milieu: Chemical-Induced Denaturation of Myoglobin is Determined by the Extent of Heme Dissociation on Interaction with Crowders.

Generally, in vivo function and structural changes are studied by probing proteins in a dilute solution under in vitro conditions, which is believed to be mimicking proteins in intracellular milieu. Earlier, thermal-induced denaturation of myoglobin, in the milieu of crowder molecule showed destabilization of the metal protein. Destabilization of protein by thermal-induced denaturation involves a large extrapolation, so, the reliability is questionable. This led us to measure the effects of macromolecular crowding on its stability by chemical-induced denaturation of the protein using probes like circular dichroism and absorption spectroscopy in the presence of dextran 70 and ficoll 70 at various pHs (acidic: 6.0, almost neutral:7.0 and basic: 8.0). Observations showed that the degree of destabilization of myoglobin was greater due to ficoll 70 as compared to that of dextran 70 so it can be understood that the nature of the crowder or the shape of the crowder has an important role towards the stability of proteins. Additionally, the degree of destabilization was observed as pH dependent, however the pH dependence is different for different crowders. Furthermore, isothermal titration calorimetry and molecular docking studies confirmed that both the crowders (ficoll and dextran) bind to heme moiety of myoglobin and a single binding site was observed for each.

The anti-oxidant enzyme, Prdx6 might have cis-acting regulatory sequence(s).

Peroxiredoxin 6 (Prdx6) is a ubiquitously expressed 1-cysteine Peroxiredoxin found throughout all phyla. In mammals, under different physiological conditions, it has evolved from a peroxidase to a multifunctional enzyme. Among the mammalian Prdx6's, human and rat Prdx6's are the most extensively studied. Our study revealed that human and rat Prdx6's exhibit differences in their peroxidase activity. These two Prdx6's have only 8% difference in their primary sequence (with 19 amino acids) with no apparent modification at any of the key conserved residues. In the present communication, we have investigated the roles of thermodynamics, structure and internal flexibility of Prdx6 to account for the difference in their peroxidase activity. We discovered that these amino acid variations have led to structural alterations in human Prdx6 so that it shows enhanced intrinsic dynamics (or flexibility) than the rat protein. We could also identify the gain of intrinsic dynamics of the catalytic site in human Prdx6 due to relocation of an important active site residue (R132) to the loop region as the most plausible reason for high catalytic activity in the human protein as compared to rat variant. Since it is the thioredoxin fold that upholds the peroxidase function, certain structural alteration in the Prdx6 structure might help to regulate the efficiency of thioredoxin folds. Our results hint that Prdx6 might have a cis-acting regulatory sequence(s).

Formation of molten globule state in horse heart cytochrome c under physiological conditions: Importance of soft interactions and spectroscopic approach in crowded milieu.

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.

Ionic Liquid Green Assembly-Mediated Migration of Piperine from Calf-Thymus DNA: A New Possibility of the Tunable Drug Delivery System.

Biocompatible surface-active ionic liquid (SAIL) was used first to study the deintercalation process of a well-known natural compound piperine (PIP) as an anticancer drug, obtained from PIP-calf thymus DNA (ctDNA) complex under controlled experimental conditions. In this study, we have been exploring the interaction of PIP in SAIL (1-butyl-3-methylimidazolium octyl sulfate ionic liquid ([C4mim][C8OSO3])), ctDNA, and deintercalation of PIP from the PIP-ctDNA complex through SAIL micelle using various spectroscopic techniques. Absorption, emission, and lifetime decay measurements provide strong evidence of the relocation of PIP molecules from ctDNA to SAIL micelle. Fluorescence quenching and steady-state fluorescence anisotropy were employed to examine the exact location of PIP in different media. Moreover, the surface tension technique was also employed to confirm the release of PIP molecules from the PIP-ctDNA complex in the presence of SAIL. Circular dichroism analysis suggested that SAIL micelle does not perturb the ctDNA structure, which supported the fact that SAIL micelle can be used as a safe vehicle for PIP. Overall, the study highlighted a novel strategy for deintercalation of drug using SAIL because the release of the drug can be controlled over a period by varying the concentration and composition of the SAIL.

First evidence of formation of pre-molten globule state in myoglobin: A macromolecular crowding approach towards protein folding in vivo.

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.