Identification of potential molecular targets and repurposed drugs for tuberculosis using network-based screening approach, molecular docking, and simulation.

The spread of drug-resistant strains of tuberculosis has hampered efforts to control the disease worldwide. The Mycobacterium tuberculosis cell wall envelope is dynamic, with complex features that protect it from the host immunological response. As a result, the bacterial cell wall components represent a potential target for drug discovery. Protein-protein interaction networks (PPIN) are critical for understanding disease conditions and identifying precise therapeutic targets. We used a rational theoretical approach by constructing a PPIN with the proteins involved in cell wall biosynthesis. The PPIN was constructed through the STRING database and embB was identified as a key protein by using four topological measures, betweenness, closeness, degree, and eigenvector, in the CytoNCA tool in Cytoscape. The 'Drug repurposing' approach was employed to find suitable inhibitors against embB. We used the Schrödinger suites for molecular docking, molecular dynamics simulation, and binding free energy calculations to validate the binding of protein with the ligand. FDA-approved drugs from the ZINC database and DrugBank were screened against embB (PDB ID: 7BVF) using high-throughput virtual screening, standard precision, and extra precision docking. The drugs were screened based on the XP docking score of the standard drug ethambutol. Accordingly, from the top five hits, azilsartan and dihydroergotamine were selected based on the binding free energy values and were further subjected to Molecular Dynamics Simulation studies for 100 ns. Our study confirms that Azilsartan and Dihydroergotamine form stable complexes with embB and can be used as potential lead molecules based on further in vitro and in vivo experimental validation.Communicated by Ramaswamy H. Sarma.

Investigation of Molecular Interactions Mechanism of Pembrolizumab and PD-1.

Human programmed cell death protein 1 (PD-1) is a checkpoint protein involved in the regulation of immune response. Antibodies are widely used as inhibitors that block the immune checkpoint, preventing strong immune responses. Pembrolizumab is an FDA-approved IgG4 antibody with PD-1 inhibitory ability for the treatment of melanoma. In this study, we investigated the effect of Pembrolizumab on the conformational changes in PD-1 using extensive molecular modeling and simulation approaches. Our study revealed that during the 200 ns simulation, the average values of the solvent accessible surface area, the radius of gyration, and internal hydrogen bonds of PD-1 were 64.46 nm2, 1.38 nm and 78, respectively, while these values of PD-1 in the PD-1/Pembrolizumab complex were 67.29 nm2, 1.39 nm and 76, respectively. The RMSD value of PD-1 gradually increased until 80 ns and maintained its stable conformation at 0.32 nm after 80 ns, while this value of PD-1 in the PD-1/Pembrolizumab complex maintained an increasing trend during 200 ns. The interaction between PD-1 and Pembrolizumab led to a flexible but stable structure of PD-1. PD-1 rotated around the rotation axis of the C'D loop and gradually approached Pembrolizumab. The number of hydrogen bonds involved in the interactions on the C and C' strands increased from 4 at 100 ns to 7 at 200 ns. The strong affinity of Pembrolizumab for the C'D and FG loops of PD-1 disrupted the interactions between PD-1 and PD-L1. Inhibition of the interaction between PD-1 and PD-L1 increased the T cell activity, and is effective in controlling and curing cancer. Further experimental work can be performed to support this finding.

Conserved acidic second shell residue modulates the structure, stability and activity of non-seleno human peroxiredoxin 6.

1-Cys peroxiredoxin6 (Prdx6) is unique and inducible bifunctional enzyme in the mammalian lungs and plays a role in the progression and inhibition of cancerous cells at different stages. The enzyme possesses two distinct active sites for phospholipase A2 and peroxidase activity. The conserved residues surrounding the peroxidase active site, also called as second shell residues are Glu50, Leu71, Ser72, His79 and Arg155. Since there is no study done about the active site stabilization of the transition state of Prdx6, there are a lot of questions unanswered regarding the Prdx6 peroxidase activity. In order to evaluate the role of second shell conserved residue Glu50, present in close vicinity to peroxidatic active site, we substituted this negatively charged residue with Alanine and Lysine. To explore the effect of mutation on the biophysical parameters, the mutant proteins were compared with Wild-Type by using biochemical, biophysical, and in silico methods. Comparative spectroscopic methods and enzyme activity demonstrate that the Glu50 plays a significant role in maintaining the structure, stability, and function of protein. From the results we conclude that Glu50 significantly controls the structure; stability and may be involved in the active site stabilization of transition state for proper position of diverse peroxides.

Synthesis of Thermo-Responsive Monofunctionalized Diblock Copolymer Worms.

Poly(glycerol monomethacrylate)-block-poly(2-hydroxypropyl methacrylate) (PGMA-PHPMA) with worm-like morphology is a typical example of reversible addition-fragmentation chain transfer (RAFT) dispersion polymerized thermo-responsive copolymer via polymerization-induced self-assembly (PISA) in aqueous solution. Chain transfer agents (CTAs) are the key component in controlling RAFT, the structures of which determine the end functional groups of the polymer chain. It is therefore of interest to monofunctionalize the polymers via CTA moiety, for bioactive functionality conjugation and in the meantime maintain the precisely controlled morphology of the copolymers and the related property. In this work, a newly designed CTA 5-(2-(tert-butoxycarbonylamino) ethylamino)-2-cyano-5-oxopentan-2-yl benzodithioate (t-Boc CPDB) was synthesized and used for the RAFT polymerization of PGMA45-PHPMA120. Subsequently, PGMA45-PHPMA120 copolymers with primary amine, maleimide, and reduced L-glutathione (a tripeptide) monofunctionalized terminals were synthesized via deprotection and conjugation reactions. These monofunctionalized copolymers maintain worm-like morphology and thermo-responsive property in aqueous solution (10% w/v), as confirmed by the transmission electron microscopy (TEM) images, and the observation of the phase transition behavior in between 4 °C and room temperature (~20 °C), respectively. Summarily, a range of thermo-responsive monofunctionalized PGMA45-PHPMA120 diblock copolymer worms were successfully synthesized, which are expected to offer potential biomedical applications, such as in polymer therapeutics, drug delivery, and diagnostics.

Withania somnifera (L.) Dunal (Ashwagandha) for the possible therapeutics and clinical management of SARS-CoV-2 infection: Plant-based drug discovery and targeted therapy.

Coronavirus disease 2019 (COVID-19) pandemic has killed huge populations throughout the world and acts as a high-risk factor for elderly and young immune-suppressed patients. There is a critical need to build up secure, reliable, and efficient drugs against to the infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. Bioactive compounds of Ashwagandha [Withania somnifera (L.) Dunal] may implicate as herbal medicine for the management and treatment of patients infected by SARS-CoV-2 infection. The aim of the current work is to update the knowledge of SARS-CoV-2 infection and information about the implication of various compounds of medicinal plant Withania somnifera with minimum side effects on the patients' organs. The herbal medicine Withania somnifera has an excellent antiviral activity that could be implicated in the management and treatment of flu and flu-like diseases connected with SARS-CoV-2. The analysis was performed by systematically re-evaluating the published articles related to the infection of SARS-CoV-2 and the herbal medicine Withania somnifera. In the current review, we have provided the important information and data of various bioactive compounds of Withania somnifera such as Withanoside V, Withanone, Somniferine, and some other compounds, which can possibly help in the management and treatment of SARS-CoV-2 infection. Withania somnifera has proved its potential for maintaining immune homeostasis of the body, inflammation regulation, pro-inflammatory cytokines suppression, protection of multiple organs, anti-viral, anti-stress, and anti-hypertensive properties. Withanoside V has the potential to inhibit the main proteases (Mpro) of SARS-CoV-2. At present, synthetic adjuvant vaccines are used against COVID-19. Available information showed the antiviral activity in Withanoside V of Withania somnifera, which may explore as herbal medicine against to SARS-CoV-2 infection after standardization of parameters of drug development and formulation in near future.

Addition of Mercury Causes Quenching of NIR Fluorescence Emission Spectra of a Photoactivatable PAiRFP1 Protein.

BACKGROUND: Biliverdin (BV) containing far-red light photoactivatable near-infrared fluorescent protein (NIR-FP) named PAiRFP1 has been developed by directed molecular evolution from one bathy bacteriophytochrome of Agrobacterium tumefaciens C58 called Agp2 or AtBphP2. Usually, the fluorescence intensity of the NIR emission spectra of PAiRFP1 tends to increase upon repeated excitation by far-red light. OBJECTIVE: This study aimed at exploring the role of PAiRFP1 and its mutants, such as V386A, V480A, and Y498H, as NIR biosensors for the detection of Hg2+ ions in the buffer solutions. METHODS: In this study, we used PCR-based site-directed reverse mutagenesis, fluorescence spectroscopy, and molecular modeling approaches on PAiRFP1 and its mutants. RESULTS: It was found that PAiRFP1 and its mutants experienced strong quenching of NIR fluorescence emission spectra upon the addition of different concentrations (0-3µM) of mercuric chloride (HgCl2). CONCLUSION: We hypothesized that PAiRFP1 and its variants have some potential to be used as NIR biosensors for the in vitro detection of Hg2+ ions in biological media. Moreover, we also hypothesized that PAiRFP1 would be the best tool to use as a NIR biosensor to detect Hg2+ ions in living organisms because of its higher signal-to-noise (SNR) ratio than other infra-red fluorescent proteins.

The Molecular Basis of the Effect of Temperature on the Structure and Function of SARS-CoV-2 Spike Protein.

The remarkable rise of the current COVID-19 pandemic to every part of the globe has raised key concerns for the current public healthcare system. The spike (S) protein of SARS-CoV-2 shows an important part in the cell membrane fusion and receptor recognition. It is a key target for vaccine production. Several researchers studied the nature of this protein under various environmental conditions. In this work, we applied molecular modeling and extensive molecular dynamics simulation approaches at 0°C (273.15 K), 20°C (293.15 K), 40°C (313.15 K), and 60°C (333.15 K) to study the detailed conformational alterations in the SARS-CoV-2 S protein. Our aim is to understand the influence of temperatures on the structure, function, and dynamics of the S protein of SARS-CoV-2. The structural deviations, and atomic and residual fluctuations were least at low (0°C) and high (60°C) temperature. Even the internal residues of the SARS-CoV-2 S protein are not accessible to solvent at high temperature. Furthermore, there was no unfolding of SARS-CoV-2 spike S reported at higher temperature. The most stable conformations of the SARS-CoV-2 S protein were reported at 20°C, but the free energy minimum region of the SARS-CoV-2 S protein was sharper at 40°C than other temperatures. Our findings revealed that higher temperatures have little or no influence on the stability and folding of the SARS-CoV-2 S protein.

Effect of polyol osmolytes on the structure-function integrity and aggregation propensity of catalase: A comprehensive study based on spectroscopic and molecular dynamic simulation measurements.

Owing to the ability of catalase to function under oxidative stress vis-à-vis its industrial importance, the structure-function integrity of the enzyme is of prime concern. In the present study, polyols (glycerol, sorbitol, sucrose, xylitol), were evaluated for their ability to modulate structure, activity and aggregation of catalase using in vitro and in silico approaches. All polyols were found to increase catalase activity by decreasing Km and increasing Vmax resulting in enhanced catalytic efficiency (kcat/Km) of the enzyme. Glycerol was found to be the most efficient polyol with a kcat/Km increase from 4.38 × 104 mM-1 S-1 (control) to 5.8 × 105 mM-1 S-1. Correlatively with this, enhanced secondary structure with reduced hydrophobic exposure was observed in all polyols. Furthermore, increased stability, with an increase in melting temperature by 15.2 °C, and almost no aggregation was observed in glycerol. Overall, ability to regulate structure-function integrity and aggregation propensity was highest for glycerol and lowest for xylitol. Simulation studies were performed involving structural dynamics measurement, principal component analysis and free energy landscape analysis. Altogether, all polyols were stabilizing in nature and glycerol, in particular, has potential to efficiently prevent not only the aggregation of the antioxidant defense system but might also serve as a stability aid during industrial processing of catalase.

Investigating the binding mechanism of topiramate with bovine serum albumin using spectroscopic and computational methods.

Various spectroscopic techniques involving fluorescence spectroscopy, circular dichroism (CD), and computational approaches were used to elucidate the molecular aspects of interaction between the antiepileptic drug topiramate and the multifunctional transport protein bovine serum albumin (BSA) under physiological conditions. Topiramate quenched BSA fluorescence in a static quenching mode, according to the Stern-Volmer quenching constant (Ksv ) data derived from fluorescence spectroscopy for the topiramate-BSA complex. The binding constant was also used to calculate the binding affinity for the topiramate-BSA interaction. Fluorescence and circular dichroism experiments demonstrate that the protein's tertiary structure is affected by the microenvironmental alterations generated by topiramate binding to BSA. To establish the exact binding site, interacting residues, and interaction forces involved in the binding of topiramate to BSA, molecular modeling and simulation approaches were used. According to the Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) calculations, the average binding energy between topiramate and BSA is -421.05 kJ/mol. Topiramate was discovered to have substantial interactions with BSA, changing the structural dynamic and Gibbs free energy landscape patterns.

In silico prediction of natural compounds as potential multi-target inhibitors of structural proteins of SARS-CoV-2.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a colossal loss to human health and lives and has deeply impacted socio-economic growth. Remarkable efforts have been made by the scientific community in containing the virus by successful development of vaccines and diagnostic kits. Initiatives towards drug repurposing and discovery have also been undertaken. In this study, we compiled the known natural anti-viral compounds using text mining of the literature and examined them against four major structural proteins of SARS-CoV-2, namely, spike (S) protein, nucleocapsid (N) protein, membrane (M) protein and envelope (E) protein. Following computational approaches, we identified fangchinoline and versicolactone C as the compounds to exhibit strong binding to the target proteins and causing structural deformation of three structural proteins (N, S and M). We recommend the inhibitory effects of these compounds from our study should be experimentally validated against SARS-CoV-2.Communicated by Ramaswamy H. Sarma.

High-resolution MD Simulation Studies to Get Mechanistic Insights into the Urea-induced Denaturation of Human Sphingosine Kinase 1.

BACKGROUND: Sphingosine kinase 1 (SPhK1) is a crucial signaling enzyme involved in cell proliferation, cellular survival, stimulation of angiogenesis, and apoptosis prevention. Recently, we have reported the unfolding kinetics of SPhK1 using molecular dynamics (MD) simulation, circular dichroism, and fluorescence spectroscopy. We found that SPhK1 showed a biphasic unfolding with an intermediate state (~ 4.0 M urea). OBJECTIVE: We aim to understand the impact of MD simulation duration on the structure, function, and dynamics of proteins. In order to get deeper insights into the folding mechanism, an extended MD simulation is required. METHODS: Here, we extended the MD simulations time scale from 100 to 300 ns on SPhK1 at increasing urea concentration to explore structural changes in the SPhK1. RESULTS: The results suggested a constant form of the unfolding of SPhK1 upon extending the simulation time scale at different urea concentrations. Furthermore, we showed step by step unfolding and percentage of secondary structure contents in SPhK1 under the influence of urea at each concentration. CONCLUSION: The results from the current work revealed a uniform pattern of the SPhK1 unfolding at different urea concentrations. This study provides deeper mechanistic insights into the urea-induced denaturation of SPhK1.

Remdesivir Strongly Binds to RNA-Dependent RNA Polymerase, Membrane Protein, and Main Protease of SARS-CoV-2: Indication From Molecular Modeling and Simulations.

Development of new drugs is a time-taking and expensive process. Comprehensive efforts are being made globally toward the search of therapeutics against SARS-CoV-2. Several drugs such as remdesivir, favipiravir, ritonavir, and lopinavir have been included in the treatment regimen and shown effective results in several cases. Among the existing broad-spectrum antiviral drugs, remdesivir is found to be more effective against SARS-CoV-2. Remdesivir has broad-spectrum antiviral action against many single-stranded RNA viruses including pathogenic SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). In this study, we proposed that remdesivir strongly binds to membrane protein (Mprotein), RNA-dependent RNA polymerase (RDRP), and main protease (Mprotease) of SARS-CoV-2. It might show antiviral activity by inhibiting more than one target. It has been found that remdesivir binds to Mprotease, Mprotein, and RDRP with -7.8, -7.4, and -7.1 kcal/mol, respectively. The structure dynamics study suggested that binding of remdesivir leads to unfolding of RDRP. It has been found that strong binding of remdesivir to Mprotein leads to decrease in structural deviations and gyrations. Additionally, the average solvent-accessible surface area of Mprotein decreases from 127.17 to 112.12 nm2, respectively. Furthermore, the eigenvalues and the trace of the covariance matrix were found to be low in case of Mprotease-remdesivir, Mprotein-remdesivir, and RDRP-remdesivir. Binding of remdesivir to Mprotease, Mprotein, and RDRP reduces the average motions in protein due to its strong binding. The MMPBSA calculations also suggested that remdesivir has strong binding affinity with Mprotein, Mprotease, and RDRP. The detailed analysis suggested that remdesivir has more than one target of SARS-CoV-2.

Impact of amino acid substitutions on the behavior of a photoactivatable near infrared fluorescent protein PAiRFP1.

A photoactivatable near-infrared fluorescent protein (NIR-FP) PAiRFP1 has been developed by 15 amino acid substitutions in its nonfluorescent template Agp2. In our previous communication, we investigated the role of three amino acids in PHY domain distal from BV molecule. The impact of the twelve amino acids in GAF domain, especially five residues near BV-binding pocket is unclear. In this paper, PCR based reverse mutagenesis, spectroscopic methods, molecular modelling and simulations have been employed to explore the roles of these substitutions during the molecular evolution of PAiRFP1. It was found that the residue L163 is important for protein folding in PAiRFP1. The residues F244 and C280 exerted remarkable effects on molar extinction coefficient, NIR fluorescence quantum yield, molecular brightness, fluorescence fold, and dark recovery rate. The residues F244 and V276 modulate the maximum absorption and emission peak position. The reverse mutant L168M exhibited a higher fluorescence fold than PAiRFP1. Additionally, the reverse mutants V203A, V294E, S218G and D127G possessed better spectral properties than PAiRFP1. This study is important for the rational design of a better BphP-based photoactivatable NIR-FPs.

Pyrazoline analogs as potential anticancer agents and their apoptosis, molecular docking, MD simulation, DNA binding and antioxidant studies.

N-formyl pyrazoline derivatives (3a-3l) were designed and synthesized via Michael addition reaction through cyclization of chalcones with hydrazine hydrate in presence of formic acid. The structural elucidation of N-formyl pyrazoline derivatives was carried out by various spectroscopic techniques such as 1H, 13C NMR, FT-IR, UV-visible spectroscopy, mass spectrometry and elemental analysis. Anticancer activity of the pyrazoline derivatives (3a-3l) was evaluated against human lung cancer (A549), fibrosarcoma cell lines (HT1080) and human primary normal lung cells (HFL-1) by MTT assay. The results of anticancer activity showed that potent analogs 3b and 3d exhibited promising activity against A549 (IC50 = 12.47 ± 1.08 and 14.46 ± 2.76 µM) and HT1080 (IC50 = 11.40 ± 0.66 and 23.74 ± 13.30 µM) but low toxic against the HFL-1 (IC50 = 116.47 ± 43.38 and 152.36 ± 22.18 µM). The anticancer activity of potent derivatives (3b and 3d) against A549 cancer cell line was further confirmed by flow cytometry based approach. DNA binding interactions of the pyrazoline derivatives 3b and 3d have been carried out with calf thymus DNA (Ct-DNA) using absorption, fluorescence and viscosity measurements, circular dichroism and cyclic voltammetry. Antioxidant potential of N-formyl pyrazoline derivatives (3a-3l) has been also estimated through DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical and H2O2. Results revealed that all the compounds exhibited significant antioxidant activity. In silico molecular modelling and ADMET properties of pyrazoline derivatives were also studied using PyRx software against topoisomerase II receptor with PDB ID: 1ZXM to explore their best hits. MD simulation of 3b and 3d was also carried out with topoisomerase II for structure-function correlation in a protein. HuTopoII inhibitory activity of the analogs (3a-3l) was examined by relaxation assay at varying concentrations 100-1000 µM.

In Silico Studies on Psilocybin Drug Derivatives Against SARS-CoV-2 and Cytokine Storm of Human Interleukin-6 Receptor.

Various metabolites identified with therapeutic mushrooms have been found from different sources and are known to have antibacterial, antiviral, and anticancer properties. Over thousands soil growth-based mushroom metabolites have been discovered, and utilized worldwide to combat malignancy. In this study, psilocybin-mushroom that contains the psychedelic compounds such as psilacetin, psilocin, and psilocybine were screened and found to be inhibitors of SARS-CoV-2 Mprotease. It has been found that psilacetin, psilocin, and psilocybine bind to Mprotease with -6.0, -5.4, and -5.8 kcal/mol, respectively. Additionally, the psilacetin was found to inhibit human interleukin-6 receptors to reduce cytokine storm. The binding of psilacetin to Mprotease of SARS-CoV-2 and human interleukin-6 receptors changes the structural dynamics and Gibbs free energy patterns of proteins. These results suggested that psilocybin-mushroom could be utilized as viable potential chemotherapeutic agents for SARS-CoV-2.

Mechanism of pH-induced conformational changes in MurE ligase obtained from Salmonella enterica serovar Typhi.

Communicated by Ramaswamy H. Sarma.

Comparative Analysis of Bacteriophytochrome Agp2 and Its Engineered Photoactivatable NIR Fluorescent Proteins PAiRFP1 and PAiRFP2.

Two photoactivatable near infrared fluorescent proteins (NIR FPs) named "PAiRFP1" and "PAiRFP2" are formed by directed molecular evolution from Agp2, a bathy bacteriophytochrome of Agrobacterium tumefaciens C58. There are 15 and 24 amino acid substitutions in the structure of PAiRFP1 and PAiRFP2, respectively. A comprehensive molecular exploration of these bacteriophytochrome photoreceptors (BphPs) are required to understand the structure dynamics. In this study, the NIR fluorescence emission spectra for PAiRFP1 were recorded upon repeated excitation and the fluorescence intensity of PAiRFP1 tends to increase as the irradiation time was prolonged. We also predicted that mutations Q168L, V244F, and A480V in Agp2 will enhance the molecular stability and flexibility. During molecular dynamics (MD) simulations, the average root mean square deviations of Agp2, PAiRFP1, and PAiRFP2 were found to be 0.40, 0.49, and 0.48 nm, respectively. The structure of PAiRFP1 and PAiRFP2 were more deviated than Agp2 from its native conformation and the hydrophobic regions that were buried in PAiRFP1 and PAiRFP2 core exposed to solvent molecules. The eigenvalues and the trace of covariance matrix were found to be high for PAiRFP1 (597.90 nm2) and PAiRFP2 (726.74 nm2) when compared with Agp2 (535.79 nm2). It was also found that PAiRFP1 has more sharp Gibbs free energy global minima than Agp2 and PAiRFP2. This comparative analysis will help to gain deeper understanding on the structural changes during the evolution of photoactivatable NIR FPs. Further work can be carried out by combining PCR-based directed mutagenesis and spectroscopic methods to provide strategies for the rational designing of these PAiRFPs.

Mechanistic insights into the urea-induced denaturation of human sphingosine kinase 1.

Sphingosine kinase 1 (SphK1) plays a significant role in various cellular processes, including cell proliferation, apoptosis, and angiogenesis. SphK1 is considered as an attractive target for drug development owing to its connection with several diseases, including cancer. In the current work, the urea-induced unfolding of SphK1 was performed at pH 8.0 and 25 °C using CD and fluorescence spectroscopy. SphK1 follows a biphasic unfolding transition (N â‡Œ I â‡Œ D) with an intermediate (I) state populated around 4.0 M urea concentration. The circular dichroism ([θ]222) and fluorescence emission spectra (λmax) of SphK1 with increasing concentrations of urea were analyzed to calculate Gibbs free energy (ΔG0) for both the transitions (N â‡Œ I and I â‡Œ D). A significant overlap of both the transitions obtained by two spectroscopic properties ([θ]222 and λmax) was observed, indicating that both N â‡Œ I and I â‡Œ D transition follow two-step equilibrium unfolding pattern. Also, we performed 100 ns molecular dynamics (MD) simulations to get atomistic insights into the structural changes in SphK1 with increasing urea concentrations. Our results showed a consistent pattern of the SphK1 unfolding with increasing urea concentrations. Together, spectroscopic and MD simulation findings provide deep insights into the unfolding mechanism and conformational features of SphK1.

Mechanistic insights into the urea-induced denaturation of a non-seleno thiol specific antioxidant human peroxiredoxin 6.

Peroxiredoxin 6 (Prdx6) is a unique enzyme among mammalian peroxiredoxins as it lacks resolving cysteine. It is found to be involved in number of different diseases including tumours and its expression level is highest in lungs as compared to other organs. It has been found that Prdx6 plays a significant role different metabolic diseases, ocular damage, neurodegeneration and male infertility. It is a bifunctional protein having phospholipase A2 and peroxidase (also has the ability to reduce phospholipid hydroperoxides) activities. In order to complete the peroxidise reaction cycle it requires glutathione catalyzed by glutathione S-transferase. Equilibrium unfolding and conformational stability of Prdx6 was studied by using urea as a chemical denaturant to understand the changes it goes under cellular stress conditions. Three different spectroscopic methods were employed to monitor urea-induced denaturation. From the results obtained, it was found that the urea denaturation of Prdx6 follows a variable two state process due to non-coincidence of the normalized transition curves obtained from different optical probes. The different denaturation curves were normalized and thermodynamic parameters, ΔGDo, Gibbs free energy change related to the urea-induced denaturation, midpoint of denaturation (Cm), and m = (δΔGD / [urea]) were obtained. The structural information of Prdx6 were further analysed by several parameters obtained by 100 ns MD simulation. The results of MD simulation clearly favour the outcome of spectroscopic studies.