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StatementThe authors have withdrawn their manuscript owing to the authors decision of withdrawing of the manuscript until all experiments are completed. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.

Four MOF-LIC-1 MOFs following the PSM strategy were prepared, characterized, and examined for the sorption of U(vi) and Th(iv). Also the nature of U(vi) interactions on MOFs was also investigated by applying DFT calculations.

Clupisoma garua is a medium-sized freshwater catfish with very high market value in Indian sub-continent. Besides immense food value, the fish is also known as a game fish. The fish samples for this study were randomly collected from middle stretch of the river Ganga at Allahabad during July 2014–June 2015. The size composition of the samples varied from 135 to 446 mm. The maximum exploitation was observed in 171–210 mm size group (29.04%), while minimum in 411–450 mm (0.83%) in stock samples. In general, lower size groups were highly exploited compared to middle and higher size groups. Male population dominates over females in 211–250, 251–290 and 331–370 mm size groups, in contrary, female was dominant in size groups from 131–170, 171–210 and 291–330 mm.

We report the NMR resonance assignments of N-terminal signal sequence deleted secretory protein Rv0603 (∆1–28−Rv0603) from Mycobacterium tuberculosis H37Rv. ∆1–28−Rv0603 displayed good peak yield and signal dispersion in 2D [15N-1H] HSQC spectrum, which prompted us to proceed for resonance assignments on this construct. Standard triple-resonance experiments for resonance assignments were recorded on [U-15N]-∆Rv0603 and [U-15N, 13C]-∆Rv0603 samples. We obtained 97% of backbone 1HN, 98% of 13Cα, 98% of 1Hα, 96% of 13C´, 100% of 13Cβ, 100% of 1Hβ and 98% of side-chain 1H chemical shifts. This protein does not show any sequence similarity to any other protein of known structure. Determination of its solution structure would facilitate understanding of its biological function.

A series of functionalized metal organic frameworks (MOFs) were synthesized by the post-synthetic modification (PSM) of Zr(IV)-containing UiO-66-NH2 MOFs using covalent grafting with various functional groups utilizing pendant –NH2 moieties. The tethering of amide (with/without pendant carboxylic acid), iminopyridine, phoshinic amide and sulphur-containing functionalities produced a library of eight different UiO-66-NH2 derivatives. The functionalized MOFs were characterized by FT-IR spectroscopy, NMR, PXRD, TGA, SEM-EDX and BET surface area analysis. Uranyl ion extraction with the functionalized MOFs was investigated in acidic/neutral/basic conditions (pH 1 to 9). This work presents a comprehensive study of different functionalized MOFs to investigate the effects of various analytical parameters, including pH, contact time, and desorption process. The MOFs as solid phase extractants (SPEs) provide a direct comparison of the sorption efficiencies of different functional groups on a common solid support. A phosphorous-functionalized material, UiO-66-PO-Ph, with enhanced thermal stability (∼500 °C) exhibits the best sorption capacity (∼96%) in an acidic medium (pH 3). The parent MOF UiO-66-NH2 (92%) and iminopyridine-functionalized UiO-66-IMP (90%) showed excellent sorption in neutral conditions (pH 7). Amide-containing MOFs UiO-66-AM1 (40%), UiO-66-AMMal (31%) and UiO-66-AMGlu (70%), sulfur-based MOFs UiO-66-SMA (65%) and UiO-66-SSA (27%), and phosphorous-functionalized UiO-66-PO-OPh (50%) displayed maximum sorption in basic conditions (pH 8). The kinetics studies revealed rapid uranium sorption in about 2 h due to the effective binding of uranyl ions with the anchored functional groups of MOFs; quantitative elution of uranyl ions from the MOF framework was carried out with 0.1/0.01 M HNO3. The MOFs also exhibit moderate recyclability for uranium sorption and can be regenerated by an acidic solution. The functionalized MOFs alter the stability in acidic/basic media; thus, UiO-66-NH2 is a versatile MOF material employed as an SPE for the extraction of radionuclides from aqueous media. This work also provides a platform for the development of new functionalized MOF materials for the efficient sorption of uranium as well as moderate recyclability for its removal, and the potential applications include the removal of uranium from aqueous waste streams.

The availability of complete genome sequence of Mycobacterium tuberculosis has provided an important tool to understand the mycobacterial biology with respect to host-pathogen interaction, which is an unmet need of the hour owing to continuous increasing drug resistance. Hypothetical proteins are often an overlooked pool though half the genome encodes for such proteins of unknown function that could potentially play vital roles in mycobacterial biology. In this context, we report the structural and functional characterization of the hypothetical protein Rv3272. Sequence analysis classifies Rv3272 as a Family III CoA transferase with the classical two domain structure and conserved Aspartate residue (D175). The crystal structure of the wild type protein (2.2 Å) demonstrated the associated inter-locked dimer while that of the D175A mutant co-crystallized with octanoyl-CoA demonstrated relative movement between the two domains. Isothermal titration calorimetry studies indicate that Rv3272 binds to fatty acyl-CoAs of varying carbon chain lengths, with palmitoyl-CoA (C16:0) exhibiting maximum affinity. To determine the functional relevance of Rv3272 in mycobacterial biology, we ectopically expressed Rv3272 in M. smegmatis and assessed that its expression encodes significant alteration in cell surface with marked differences in triacylglycerol accumulation. Additionally, Rv3272 expression protects mycobacteria from acidic, oxidative and antibiotic stress under in vitro conditions. Taken together, these studies indicate a significant role for Rv3272 in host-pathogen interaction.

A simple and efficient route to develop various novel functionalized MOF materials for rapid and excellent recovery of U(VI) from aqueous medium, along with selective sensing has been demonstrated in the present study. In this connection, a set of four distinct post synthetically modified (PSM) iso-reticular metal organic frameworks were synthesized from IRMOF-3 namely, IRMOF-PC (2-pyridine carboxaldehyde), IRMOF-GA (glutaric anhydride), IRMOF-SMA (sulfamic acid), and IRMOF-DPC (diphenylphosphonic chloride) for the recovery and sensing of U(VI) from aqueous medium. The MOFs were characterized by Fourier transform infrared spectroscopy (FTIR), powder XRD, BET surface area analysis, thermogravimetric analysis (TGA), NMR (13C, 1H and 31P), Scanning Electron Microscopy (SEM), and energy dispersive X-ray spectroscopy (EDX). Among all MOFs, post synthetically modified IRMOF-SMA showed enhanced thermal stability of about 420 °C. The MOFs were investigated for U(VI) sorption studies using a batch technique. All the MOFs exhibit excellent sorption capacity towards U(VI) (>90%) and maximum uptake was observed at pH 6. Sorption capacity of MOFs have the following order; IRMOF-3-DPC (300 mg U g−1) > IRMOF-SMA (292 mg U g−1) > IRMOF-PC (289 mg U g−1) > IRMOF-GA (280 mg U g−1) > IRMOF-3 (273 mg U g−1). IRMOF-DPC shows rapid sorption of uranium within 5 min with excellent uptake of U(VI) (>99%). The desorption of U(VI) was examined with different eluents and 0.01 M HNO3 was found to be most effective. The fluorescence sensing studies of U(VI) via IRMOF-3 and its PSM MOFs revealed high sensitivity and selectivity towards U(VI) over other competing rare earth metal ions (La3+, Ce4+, Sm3+, Nd3+, Gd3+, and Eu3+), wherein IRMOF-GA displayed an impressive detection limit of 0.36 mg L−1 for U(VI).

Induction of improper folding of protein structure induces protein aggregation and related proteinopathies. These pathological conditions manifest in neurodegenerative diseases like Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), and amyotrophic lateral sclerosis (ALS). Such amyloidosis is divided into four different subtypes depending upon their causative factors. These four subtypes are majorly known as AL amyloidosis, ATTR amyloidosis, AA amyloidosis, and dialysis-related beta2-microglobulin amyloidosis (ABM2). The progressive accumulation of such amyloids into the cell leads to an aggravation of diseased conditions thereby resulting in the death of the patient. There are very few intervention strategies to delay the onset process of such disease conditions as well as their early diagnosis. Hence, symptomatic management is of paramount importance to promote quality of life in aged patients. Advancement in the area of nano-biomedical technology has led to the advent of prophylactic, diagnostic, and therapeutic applications for various disease conditions.

Klebsiella pneumoniae is a member of the ESKAPE panel of pathogens that are top priority to tackle AMR. Bacterial peptidyl tRNA hydrolase (Pth), an essential, ubiquitous enzyme, hydrolyzes the peptidyl-tRNAs that accumulate in the cytoplasm because of premature termination of translation. Pth cleaves the ester bond between 2′ or 3′ hydroxyl of the ribose in the tRNA and C-terminal carboxylate of the peptide, thereby making free tRNA available for repeated cycles of protein synthesis and preventing cell death by alleviating tRNA starvation. Pth structures have been determined in peptide-bound or peptide-free states. In peptide-bound state, highly conserved residues F67, N69 and N115 adopt a conformation that is conducive to their interaction with peptide moiety of the substrate. While, in peptide-free state, these residues move away from the catalytic center, perhaps, in order to facilitate release of hydrolysed peptide. Here, we present a novel X-ray crystal structure of Pth from Klebsiella pneumoniae (KpPth), at 1.89 A resolution, in which out of the two molecules in the asymmetric unit, one reflects the peptide-bound while the other reflects peptide-free conformation of the conserved catalytic site residues. Each molecule of the protein has canonical structure with seven stranded β-sheet structure surrounded by six α-helices. MD simulations indicate that both the forms converge over 500 ns simulation to structures with wider opening of the crevice at peptide-binding end. In solution, KpPth is monomeric and its 2D-HSQC spectrum displays a single set of well dispersed peaks. Further, KpPth was demonstrated to be enzymatically active on BODIPY-Lys-tRNALys3.

The proteins secreted by bacteria contribute to immune mediated gastric inflammation and epithelial damage; thus aid bacterial invasion in host tissue, and may also interact with host proteins, conspirating a mechanism against host-immune system. The Histone-like DNA binding protein is one of the most abundant nucleoid-associated proteins in Helicobacter pylori (H. pylori). The protein -referred here as Hup- is also secreted in vitro by H. pylori, thus it may have its role in disease pathogenesis. This is possible only if Hup interact with some human proteins including Small-Ubiquitin-like-Modifier (SUMO) proteins. Studies have established that SUMO-proteins participate in various innate-immune pathways and thus promote an efficient immune response to combat pathogenic infections. Sequence analysis revealed the presence of two SUMO interacting motifs (SIMs) and several positively charged lysine residues on the protein surface of Hup. Additionally, SUMO-proteins epitomize negatively charged surface which confers them the ability to bind to DNA/RNA binding proteins. Based on the presence of SIMs as well as charge complementarity between the proteins, it is legitimate to consider that Hup protein would bind to SUMO-proteins. The present study has been undertaken to establish this interaction for the first time using NMR in combination with ITC and other biophysical techniques.

Rv3488 of Mycobacterium tuberculosis H37Rv has been assigned to the phenolic acid decarboxylase repressor (PadR) family of transcriptional regulators that play key roles in multidrug resistance and virulence of prokaryotes. The binding of cadmium, zinc, and several other metals to Rv3488 was discovered and characterized by isothermal titration calorimetery to be an exothermic process. Crystal structures of apo-Rv3488 and Rv3488 in complex with cadmium or zinc ions were determined by X-ray crystallography. The structure of Rv3488 revealed a dimeric protein with N-terminal winged-helix-turn-helix DNA-binding domains composed of helices α1, α2, α3, and strands β1 and β2, with the dimerization interface being formed of helices α4 and α1. The overall fold of Rv3488 was similar to PadR-s2 and metal sensor transcriptional regulators. In the crystal structure of Rv3488–Cd complex, two octahedrally coordinated Cd2+ ions were present, one for each subunit. The same sites were occupied by zinc ions in the structure of Rv3488–Zn, with two additional zinc ions complexed in one monomer. EMSA studies showed specific binding of Rv3488 with its own 30-bp promoter DNA. The functional role of Rv3488 was characterized by expressing the rv3488 gene under the control of hsp60 promoter in Mycobacterium smegmatis. Expression of Rv3488 increased the intracellular survival of recombinant M. smegmatis in murine macrophage cell line J774A.1 and also augmented its tolerance to Cd2+ ions. Overall, the studies show that Rv3488 may have transcription regulation and metal-detoxifying functions and its expression in M. smegmatis increases intracellular survival, perhaps by counteracting toxic metal stress.

Leishmania donovani possess two isoforms of Rab5 (Rab5a and Rab5b), which are involved in fluid phase and receptor-mediated endocytosis, respectively. We have characterized the solution structure and dynamics of a stabilized truncated LdRab5a mutant. For the purpose of NMR structure determination, protein stability was enhanced by systematically introducing various deletions and mutations. Deletion of hypervariable C-terminal and the 20 residues LdRab5a specific insert slightly enhanced the stability, which was further improved by C107S mutation. The final construct, truncated LdRab5a with C107S mutation, was found to be stable for longer durations at higher concentration, with an increase in melting temperature by 10°C. Solution structure of truncated LdRab5a shows the characteristic GTPase fold having nucleotide and effector binding sites. Orientation of switch I and switch II regions match well with that of guanosine 5'-(β, γ-imido)triphosphate (GppNHp)-bound human Rab5a, indicating that the truncated LdRab5a attains the canonical GTP bound state. However, the backbone dynamics of the P-loop, switch I, and switch II regions were slower than that observed for guanosine 5'-(β, γ-imido)triphosphate (GMPPNP)-bound H-Ras. This dynamic profile may further complement the residue-specific complementarity in determining the specificity of interaction with the effectors. In parallel, biophysical investigations revealed the urea induced unfolding of truncated LdRab5a to be a four-state process that involved two intermediates, I1 and I2. The maximal 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (Bis-ANS) binding was observed for I2 state, which was inferred to have molten globule like characteristics. Overall, the strategy presented would have significant impact for studying other Rab and small GTPase proteins by NMR spectroscopy.

Background Twinstar is an ADF/cofilin family protein, which is expressed by the tsr gene in Drosophila melanogaster. Twinstar is one of the main regulators of actin cytoskeleton remodelling and is essential for vital cellular processes like cytokinesis and endocytosis. Methods We have characterized the structure and dynamics of Twinstar by solution NMR spectroscopy, the interaction of Twinstar with rabbit muscle actin by ITC, and biochemical activities of Twinstar through different biochemical assays using fluorescence spectroscopy and ultra-centrifugation. Results The solution structure of Twinstar shows characteristic ADF-H fold with well-formed G/F-site and F-site for interaction with actin. The structure possesses an extended F-loop, which is rigid at the base, but flexible towards its apical region. Twinstar shares similar dynamics for the G/F-site with C. elegans homologs, UNC-60A and UNC-60B. However, the dynamics of its F-loop are different from its C. elegans homologs. Twinstar shows strong affinity for ADP-G-Actin and ATP-G-Actin with Kds of ~7.6 nM and ~0.4 μM, respectively. It shows mild F-actin depolymerizing activity and stable interaction with F-actin with a Kd of ~5.0 μM. It inhibits the rate of the nucleotide exchange in a dose dependent manner. Conclusion On the basis of structure, dynamics, and biochemical activity, Twinstar can be taken to execute its biochemical role by facilitating directional growth and maintenance of length of actin filaments. General significance This study characterizes the structure, backbone dynamics, and biochemical activities of Twinstar of Drosophila, which provides an insight into the regulation of actin dynamics in the member of phylum insecta.

The ESAT-6 family proteins of Mycobacterium tuberculosis are regarded as the key mediators in mycobacterial virulence and are largely considered as antigens that can improve TB vaccines and diagnostics. We have characterized Rv3444c and Rv3445c proteins of the ESX-4 system of ESAT-6 family of M. tuberculosis H37Rv, and have experimentally established that these two proteins interact to form a heterodimeric complex. Complex formation resulted in induction of α-helical conformation and stability against chemical denaturation. To evaluate the immunogenic potential, we have immunized mice with Rv3444c or Rv3445c along with Freund's incomplete adjuvant (FIA). Immunization with Rv3444c-FIA or Rv3445c-FIA resulted in long term humoral responses. Re-stimulation of splenocytes from immunized mice resulted in significant lymphocyte proliferation with induction of TNF-α and IL-6. Further, the humoral responses to Rv3444c and Rv3445c antigens in Indian patients with active pulmonary TB (n = 44), and healthy individuals (n = 20), were investigated. Compared to healthy individuals, high levels of IgG against Rv3444c and Rv3445c were observed in TB patient's sera, indicating that these proteins are actively produced during the active phase of TB. Cellular immune responses to these proteins in active pulmonary TB patients (n = 5) were also investigated using peripheral blood mononuclear cells (PBMCs). Both the proteins induce significant lymphocyte proliferation and up-regulate the induction of TNF-α and IL-6 in TB patients.

We have studied the cucurbit[7]uril inclusion complexes of two anti-cancer drugs, palbociclib and ribociclib, in the solution and solid-state with the help of experimental and computational approaches. The formation of the cucurbit[7]uril inclusion complex, binding stoichiometry, and binding free energies in aqueous solution was determined from 1H-NMR, UV-spectroscopy and ITC studies. The binding of drugs in the ionized and unionized forms with cucurbit[7]uril was studied with the help of QTAIM analysis and free energy computations. We report the development of new co-amorphous solids of the cucurbit[7]uril inclusion complexes of drugs with enhanced thermal stability. We also report three new crystalline salt forms of palbociclib oxalate and a new dimethyl sulfoxide solvate of ribociclib succinate dihydrate in the study. The phase interrelationship between the palbociclib oxalate forms (1a, 1b, and 1c) was established from DSC and PXRD study.

Fishes are a prominent feature in most national economics and employments. Riverine ecosystems present unique opportunity to employment of fishers and youth community in the Ganga basin, India. Present study was undertaken to fish catch from the middle stretch of the Ganga river at Allahabad, India during the period July 2014 to June 2015. Annual fish landing demonstrated that the catch was dominated by miscellaneous group (22.16 kg day -1 ) followed by Oreochromis niloticus (12.76 kg day -1 ) and Sperata seenghala (12.63 kg day -1 ) and they accounted for 23.19 per cent, 13.36 per cent and 13.21 per cent, respectively. In case of Indian major carps, Cirrhinus mrigala shared maximum contribution with 7.73 kg day -1 (8.09%). Catla catla and Labeo rohita shared 1.45 (1.51%) and 2.35 kg day -1 (2.46%), respectively. The landing of fishes fluctuated from season to season from the Ganga river at Allahabad, India. For conservation point of view Cyprinus carpio and O. niloticus species should be monitored in the Ganga river. Both species are very harmful for the Ganga river ecosystem.

Actin, an ATPase superfamily protein, regulates some vital biological functions like cell locomotion, cytokinesis, synaptic plasticity and cell signaling in higher eukaryotes, and is dependent on the dynamics of actin polymerization process. Impaired regulation of actin polymerization has been implicated in the formation and deposition of rod-like paracrystalline structures called as Hirano bodies in neuronal cells of patients suffering from Alzheimer's disease, Pick's disease, Guam amyotrophic lateral sclerosis and parkinsonism-dementia complex. Aggregation of actin forming amorphous deposition in the brain cells is also associated with chronic alcoholism and aging of the neurons. In the current article, we propose the breaking of the highly amorphous and dysregulated actin aggregates using generic compounds like tetracycline, oxytetracycline, doxycycline and minocycline which are used as antibiotics against tuberculosis and infection caused due to various Gram-negative bacteria. We have investigated the effect and affinity of binding of these four compounds to that of actin aggregates using 90° light scattering, size exclusion chromatography, dynamic light scattering, circular dichroism, scanning electron microscopy, transmission electron microscopy imaging and kinetic analysis. The isothermal calorimetric measurements showed that the binding constant for the cycline family molecules used in this study range from 9.8

Rapid procedure for the separation of andrographolide from Andrographis paniculata leaves was developed to furnish a certified reference material (CRM). Andrographolide is the principle compounds, which attributed to define the quality and efficacy of A. paniculata derived medicinal products. First time, authentication, sample preparation, homogeneity, storage stability, trueness and reliability assignment by uncertainty measurement of andrographolide by high performance liquid chromatography (HPLC) were illustrated in the present paper. The impurity in the samples due to residual moisture, inorganic and organic was determined by Karl-Fisher titration, thermogravimetric analysis, and differential scanning calorimetry methods, respectively. Following the EURACHEM/CITAC guide of CG-4 statistical procedure, the certified value and corresponding expanded uncertainty of andrographolide CRM was assigned to be 99.33% ± 0.02% (k = 2) with one year stability (p

Bacterial peptidyl-tRNA hydrolase (Pth; EC 3.1.1.29) hydrolyzes the peptidyl-tRNAs accumulated in the cytoplasm and thereby prevents cell death by alleviating tRNA starvation. X-ray and NMR studies ofVibrio choleraePth (VcPth) and mutants of its key residues involved in catalysis show that the activity and selectivity of the protein depends on the stereochemistry and dynamics of residues H24, D97, N118, and N14. D97-H24 interaction is critical for activity because it increases the nucleophilicity of H24. The N118 and N14 have orthogonally competing interactions with H24, both of which reduce the nucleophilicity of H24 and are likely to be offset by positioning of a peptidyl-tRNA substrate. The region proximal to H24 and the lid region exhibit slow motions that may assist in accommodating the substrate. Helix α3 exhibits a slow wobble with intermediate time scale motions of its N-cap residue N118, which may work as a flypaper to position the scissile ester bond of the substrate. Overall, the dynamics of interactions between the side chains of N14, H24, D97, and N118, control the catalysis of substrate by this enzyme.

In order to explore the effect of ancillary aromatic rigid positional isomeric ligands and flexible aromatic dicarboxylate ligands on the architecture of coordination polymers (CPs) of Zn(II) and Cd(II) ions, employing 2,6-bis(imidazol-1-yl)pyridine (pyim2) as the main ligand, six CPs were synthesized at room temperature, namely {[Zn(pyim2)(o-BDC)]·(DMF)·(MeOH)}n (1), {[Zn2(pyim2)2(m-BDC)2]·(DMF)4}n (2), {[Zn(pyim2)(p-BDC)]·(DMF)}n (3), {[Cd(pyim2)(p-BDC)(H2O)]·(H2O)2}n (4), {[Zn(pyim2)(OBA)]·(H2O)}n (5) and {[Cd(pyim2)(OBA)]·(H2O)}n (6) (H2o-BDC = phthalic acid, H2m-BDC = isophthalic acid, H2p-BDC = terephthalic acid and H2OBA = 4,4′-oxybisbenzoic acid). Structural analysis of CPs 1–6 reveals diverse architectures and dimensionalities ranging from 1D to 3D. CP 1 possesses a 2D herringbone pleated architecture with a 44·62sql 4-connected uninodal net, while CP 2 features a 1D chain. CP 3 can be represented as a 2D parallel pleated network with a 44·62sql 4-connected uninodal net. CP 4 is a linear 1D chain with non-coordinating pendant pyim2 units. CP 5 features a right-handed 1D helical chain, resulting in a 2-fold interpenetrating 4-connected 3D CP with the 65.8 sqc-5 topology. CP 6 possesses a four-membered Cd2O4 ring forming an 8-connected 3D CP exhibiting the 424·64bcu topology. The structural variations in the CPs are primarily effected by the position, orientation and bonding fashion of the coordinated carboxylate groups as well as their flexible and rigid nature, in addition to the type of transition metal ion and orientation of the pyim2 ligands. Furthermore CPs 1–6 show thermal stability and variable photoluminescent behaviors.

Background Accumulation of toxic peptidyl-tRNAs in the bacterial cytoplasm is averted by the action of peptidyl-tRNA hydrolase (Pth), which cleaves peptidyl-tRNA into free tRNA and peptide. NMR studies are needed for a protein homolog with a complete crystal structure, for comparison with the NMR structure of Mycobacterium tuberculosis Pth. Methods The structure and dynamics of Mycobacterium smegmatis Pth (MsPth) were characterized by NMR spectroscopy and MD simulations. The thermal stability of MsPth was characterized by DSC. Results MsPth NMR structure has a central mixed seven stranded β-sheet that is enclosed by six α-helices. NMR relaxation and MD simulations studies show that most of the ordered regions are rigid. Of the substrate binding segments, the gate loop is rigid, the base loop displays slow motions, while the lid loop displays fast timescale motions. MsPth displays high thermal stability characterized by a melting temperature of 61.71 °C. Conclusion The NMR structure of MsPth shares the canonical Pth fold with the NMR structure of MtPth. The motional characteristics for the lid region, the tip of helix α3, and the gate region, as indicated by MD simulations and NMR data, are similar for MsPth and MtPth. However, MsPth has relatively less rigid base loop and more compactly packed helices α5 and α6. The packing and the dynamic differences appear to be an important contributing factor to the thermal stability of MsPth, which is significantly higher than that of MtPth. Significance MsPth structure consolidates our understanding of the structure and dynamics of bacterial Pth proteins.

Salt stress is a major environmental constraint limiting plant productivity. Tomato ( Lycopersicum esculentum) is one of the commodity vegetable that has recently been added to the list of the world's major food crops and is considered as one of the most popular vegetable. An experiment was conducted to study the effect of salicylic acid (SA) on tomato varieties grown in different levels of salt stress. Salicylic acid (SA) is a plant growth promoting compound relatively inexpensive and enhances growth and yield of crops under saline conditions. The effect of exogenous salicylic acid (25μM) application of with four NaCl concentrations (50mM,100mM, 150mM and 200mM) stressed tomato varieties was investigated. Results on some morphological parameters as plant-height and number of leaves revealed that, salt stressreduced plant-height and number of leaves. While, exogenous application of salicylic acid promoted plant-height and number of leaves and counteracted the salt stress-induced inhibition of plant-height and number of leaves.

Background In the molybdenum cofactor biosynthesis pathway, MoaA and MoaC catalyze the first step of transformation of GTP to cPMP. In M. tuberculosis H37Rv, three different genes ( Rv3111 , Rv0864 and Rv3324c ) encode for MoaC homologs. Out of these three only MoaC1 ( Rv3111 ) is secretory in nature. Methods We have characterized MoaC1 protein through biophysical, in-silico , and immunological techniques. Results We have characterized the conformation and thermodynamic stability of MoaC1, and have established its secretory nature by demonstrating the presence of anti-MoaC1 antibodies in human tuberculosis patients' sera. Further, MoaC1 elicited a dominant Th1 immune response in mice characterized by increased induction of IL-2 and IFN-γ. Conclusion Integrating these results, we conclude that MoaC1 is a structured secretory protein capable of binding with GTP and eliciting induced immune response. General significance This study would be useful for the development of vaccines against tuberculosis and to improve methods used for diagnosis of tuberculosis.

Internet of things (IOT) is the most promising area of research nowadays to reach the requirement of today’s world. This technology is persuading into our lifestyles by managing our surroundings according to our reactions and requirements. Moreover, this technology involves various electronic devices which tend to create power loss during the operation. This issue may be resolved by integrating the concept of low power digital device design technique. This low power digital design approach can be achieved through Reversible circuit design method. Here, in this paper, we have discussed about the fundamental concepts and issues of these two technologies. Further, integration of these technologies may lead to the generation of low power high efficiency IOT systems in future.

The first chapter introduces the importance of actin and its polymerization dynamics in general. It briefs about the major cellular processes that are controlled by the virtue of controlled regulation of actin polymerization process. It also summarizes different pathophysiological conditions arising due to impaired regulation of actin. Further, we also discuss the importance of acting polymerization in motility in different prokaryotic and eukaryotic cellular organisms. Finally, we conclude by introducing the phenomenon of gliding motility in apicomplexans, where actin which is the central player is tightly controlled by a minimal set of regulatory proteins. This is quite unlike higher eukaryotes where the number of similar actin-binding accessory proteins is fairly high.

A new one-dimensional double chain photoluminescent Hg(II) coordination polymer (CP), {[Hg(L)2]·(ClO4)2}n (1), was synthesized using a benzimidazole-appended tripodal tridentate ligand, 1,3,5-tris(benzimidazolylmethyl)benzene (L). The dynamic and flexible framework of 1 allows it to be entitled as first Hg(II)-based CP belonging to the rare category of CPs that exhibit multistimuli-responsive photoluminescence sensing properties and called as “smart” material. The sensitivity of this material via luminescence quenching method showing “turn off” behavior to a range of stimuli, including anions, solvents, and nitroaromatic compounds (NACs), offers more fine-grained control over its properties. 1 can easily adjust its channel dimensions to encapsulate different guest anions forming complete/partial anion-exchanged materials 1A–1B/1C–1E using NO3–, BF4–, OTf–, OTs–, and PF6– anions, respectively. Reversible (1A and 1B) and irreversible (1C–1E) anion exchange behaviors were observed for the complete and partia...

Rv3488 of

Background The GMF class of the ADF-H domain family proteins regulate actin dynamics by binding to the Arp2/3 complex and F-actin through their Site-1 and Site-2, respectively. CeGMF of C. elegans is analogous to GMFγ of human and mouse and is 138 amino acids in length. Methods We have characterized the solution structure and dynamics of CeGMF by solution NMR spectroscopy and its thermal stability by DSC. Results The solution structure of CeGMF shows canonical ADF-H fold with two additional β-strands in the β4-β5 loop region. The Site-1 of CeGMF is well formed and residues of all three regions of Site-1 show dynamic flexibility. However, the β4-β5 loop of Site-2 is less inclined towards the C-terminal, as the latter is truncated by four residues in comparison to GMF isoforms of human and mouse. Regions of Site-2 show motions on ns-ps timescale, but dynamic flexibility of β4-β5 loop is low in comparison to corresponding F-loop region of ADF/cofilin UNC-60B. A general difference in packing of α3 and α1 between GMF and ADF/cofilins was noticed. Additionally, thermal stability of CeGMF was significantly higher than its ADF/cofilin homologs. Conclusion We have presented the first solution structure of GMF from C. elegans, which highlights the structural differences between the Site-2 of CeGMF and mammalian GMF isoforms. Further, we have seen the differences in structure, dynamics, and thermal stability of GMF and ADF/cofilin. General significance This study provides a useful insight to structural and dynamics factors that define the specificity of GMF towards Arp2/3 complex.

Entity authentication and key distribution are very important cryptographic problems in mobile communication or in adhoc networks or in wireless communication at large. Mutual entity authentication is seen as the necessary process to the establishment of a secure and authentic connection. For a reliable secure communication, mutual entity authentication is very often seen as the necessity to the establishment of a secure connection. Authentication is necessary in wireless communication such as GSM or for Ad-Hoc purposes [26].Here Latin word ad-hoc means for the specific purpose only. Authentication is also necessary in situations where we need more than two entities in the authentication exchange such as a mobile user, a local AAA (Authentication, Authorization and Accounting) server and a remote (home) AAA server [8,21]. In such situations it is essential to include authentic and secure key exchange mechanism that ensures enhancement of trust in communication. This paper provides an authenticated zero knowledge protocol which is very helpful in establishing secure wireless communication in ad-hoc networks by satisfying cryptographic goals such as authentication, data integrity etc [13].

Background Bacterial peptidyl-tRNA hydrolase (Pth) is an essential enzyme that alleviates tRNA starvation by recycling prematurely dissociated peptidyl-tRNAs. The specificity of Pth for N-blocked-aminoacyl-tRNA has been proposed to be contingent upon conserved residue N14 forming a hydrogen bond with the carbonyl of the first peptide bond in the substrate. M71 is involved in forming a conserved hydrogen bond with N14. Other interactions facilitating this recognition are not known. Methods The structure, dynamics, and stability of the M71A mutant of Pth from Vibrio cholerae (VcPth) were characterized by X-ray crystallography, NMR spectroscopy, MD simulations and DSC. Results Crystal structure of M71A mutant was determined. In the structure, the dimer interface is formed by the insertion of six C-terminal residues of one molecule into the active site of another molecule. The side-chain amide of N14 was hydrogen bonded to the carbonyl of the last peptide bond formed between residues A196 and E197, and also to A71. The CSP profile of mutation was similar to that observed for the N14D mutant. M71A mutation lowered the thermal stability of the protein. Conclusion Our results indicate that the interactions of M71 with N14 and H24 play an important role in optimal positioning of their side-chains relative to the peptidyl-tRNA substrate. Overall, these interactions of M71 are important for the activity, stability, and compactness of the protein. Significance The work presented provides original and new structural and dynamics information that significantly enhances our understanding of the network of interactions that govern this enzyme's activity and selectivity.

A rigid imidazolium dicarboxylate ligand, 4,5-dicarboxy-1,3-dimethyl-1H-imidazolium iodide (H2DDII), was synthesized and employed in the construction of metal complexes or coordination polymers (CPs) comprising an anionic zwitterion (DDI−), namely, [Zn(Cl)(H2O)2(DDI)]·(H2O) (2), {[Zn2(DDI)3(H2O)2]·I}n (3), [Zn2(DDI)2I]·2H2O (4), [Zn(DDI)2(H2O)3]·(H2O)2 (5) and [Cd(DDI)2]n (6). In addition, the ligand upon crystallization forms a zwitterion, [HDDI, (1)] by losing HI and shows both intra- and intermolecular H-bonding and an interesting intermolecular CO⋯C short contact. The carboxylate unit of the ligand exhibits terminal monodentate and bidentate chelate modes of coordination. The stoichiometric ratio of metal to ligand, nature of base, type of metal salt and solvent conditions affected the architectures and dimensionality of 2–6 from zero dimensional monomers and dimers to 2D coordination polymers (CPs). Complex 2 forms a discrete molecular unit extended by supramolecular interactions to form 2D rectangular sheets. In 3, a crown shaped macrocycle results in a 2D herringbone CP and extends to a 3D supramolecular architecture on account of a coordination network containing trapped iodide as counter anions. Complex 4 forms a dimer with coordinated iodide ions and forms an infinite 1D ladder shaped supramolecular architecture. Complex 5 forms another discrete molecular unit different from 2, and extends to a 3D architecture through supramolecular interactions. Complex 6 forms a 2D CP with Cd(II) forming an interesting “fish scale pattern” and lacks any counter anion or solvent molecules. Additionally, the thermal stability of complexes 2–6 was analyzed by thermogravimetric analysis. In addition, the solid state photoluminescence properties of H2DDII and compounds 2–6 were investigated at room temperature.

An anion induced structural variation from 1D to 2D was studied for four coordination polymers (CPs) synthesized from an angular ditopic ligand 2,6-bis(imidazol-1-yl)pyridine (pyim2) under different solvent conditions, namely {[Zn(pyim2)2]·(PF6)2}n (1) (1D), {[trans-Cd(pyim2)2(DMF)2]·(PF6)2·(DMF)2}n (2) (1D), {[trans-Cd(pyim2)2(H2O)2]·(NO3)2}n (3) (2D) and {[trans-Cd(pyim2)2(H2O)2]·(PF6)·(NO3)·(H2O)2}n (4) (1D). An anion triggered single-crystal-to-single-crystal (SCSC) transformation was observed from 1D to 2D by counteranion exchange from PF6− to NO3− and vice versa. In addition, the cause for SCSC transformation involving the metal–ligand bond breaking/formation phenomenon and enhancement/decrement of the network dimensionality is discussed. The anion/solvent exchange transformations were confirmed directly through single crystal X-ray diffraction, IR spectroscopy, PXRD and elemental analysis. Furthermore, the thermal and solid state photoluminescence properties of 1–4 were analyzed.

The nematode Caenorhabditis elegans has two ADF (actin-depolymerizing factor)/cofilin isoforms, UNC-60A and UNC-60B, which are expressed by the unc60 gene by alternative splicing. UNC-60A has higher activity to cause net depolymerization, and to inhibit polymerization, than UNC-60B. UNC-60B, on the other hand, shows much stronger severing activity than UNC-60A. To understand the structural basis of their functional differences, we have determined the solution structures of UNC-60A and UNC-60B proteins and characterized their backbone dynamics. Both UNC-60A and UNC-60B show a conserved ADF/cofilin fold. The G-actin (globular actin)-binding regions of the two proteins are structurally and dynamically conserved. Accordingly, UNC-60A and UNC-60B individually bind to rabbit muscle ADP–G-actin with high affinities, with Kd values of 32.25 nM and 8.62 nM respectively. The primary differences between these strong and weak severing proteins were observed in the orientation and dynamics of the F-actin (filamentous actin)-binding loop (F-loop). In the strong severing activity isoform UNC-60B, the orientation of the F-loop was towards the recently identified F-loop-binding region on F-actin, and the F-loop was relatively more flexible with 14 residues showing motions on a nanosecond–picosecond timescale. In contrast, in the weak severing protein isoform UNC-60A, the orientation of the F-loop was away from the F-loop-binding region and inclined towards its own C-terminal and strand β6. It was also relatively less flexible with only five residues showing motions on a nanosecond–picosecond timescale. These differences in structure and dynamics seem to directly correlate with the differential F-actin site-binding and severing properties of UNC-60A and UNC-60B, and other related ADF/cofilin proteins.

Four 2D helical coordination polymers (CPs) (1–4) were synthesized using achiral 2,6-bis (imidazol-1-yl)pyridine (pyim2) ligand with metal nitrates (metal = zinc and cadmium), which showed that variation in the solvent condition leads to difference in geometry around the central metal ion and results in chiral/achiral behaviour of these CPs. By using (pyim2), [trans-Zn(pyim2)2(NO3)2]n (1) was obtained by unary solvent (MeOH), while [trans- Cd(pyim2)2(NO3)2] n (2) was formed under binary solvent mixtures (DMF/MeOH). On the other hand, in ternary solvent mixture (DMF/MeOH/H2O) it resulted into an achiral {[trans-Zn(pyim2)2(H2O)2] ·(NO3)2}n (3) and homochiral {[cis-Cd(pyim2)2(H2O)2] ·(NO3)2}n (4) coordination polymer, respectively. The homochiral behaviour of the coordination polymer (4) was further studied by solid state CD spectra and also its optical behaviour was analyzed by polarimetry.

A hexanuclear hexagonal organotin macrocycle [(n-Bu3Sn)6(μ-L)6(I–)2(MeOH)6] (1) was synthesized in a 1:1 reaction of (n-Bu3Sn)2O and 4,5-dicarboxy-1,3-dimethyl-1H-imidazol-3-ium iodide (LH2I). The molecular structure of 1 reveals that it is a 42-membered hexatin macrocycle possessing a C3 (pseudo-S6) symmetry. The alternate up–down arrangement of imidazolium units allows the molecule to assume a chair topology. The hexagonal packing of these macrocycles, in the solid-state, results in nanoscale one-dimensional channels which entrap two I– ions in close proximity (∼3.7 A) as a result of various supramolecular interactions.

The loss of biodiversity is one of the most reflective effects of humans on the global perspective and it is more and more urgent to understand how this loss will affect and what will be the profound consequences to the ecosystem functioning. Non-native fishes can cause considerable adverse impacts on the function of aquatic ecosystems and loss of biodiversity. Ecology of ecosystem focuses on the fluctuation of energy and nutrients through ecological systems. It has been confirmed that the fishes are sensitive indicators of environmental degradation and alteration. Study was undertaken during the period of October 2015 to September 2016 from the Paisuni river, India. Fish faunas of the Paisuni river have harbors of 58 species belonging to 5 order, 18 family and 43 genera. Cypriniformes and Cyprinidae were the most rich fish species order and families, respectively from the river. The Cyprinidae family has highest harbors family with 25 fish species. The family Anabaniitidae has 5 fish species which is second dominant family from the Paisuni river. According to abundance, Cyprinus carpio and Oreochromis niloticus were powerfully invaded in the Paisuni river. The detonated frequency of O. niloticus and C. carpio was recorded from the Paisuni river. Exotic species is alarming for indigenous fish species biodiversity. C. carpio and O. niloticus are frequently recorded in the Ganga river. Very highly important and ecological indicator fish species, Tor mahseer, Tor tor is declining in the catch. Current ecosystem functioning is favour to non-native species from the Paisuni river.

Background We have characterized two immunogenic proteins, Rv1197 and Rv1198, of the Esx-5 system of the ESAT-6 family of Mycobacterium tuberculosis H37Rv. Methods The complex formation between Rv1197 and Rv1198 was characterized by biophysical techniques. The reactivity of serum from TB patients towards these proteins was characterized by ELISA. Lymphocyte proliferation and cytokine induction were followed in restimulated splenocytes from immunized mice by using MTT assay and CBA flowcytometry, respectively. Results Rv1197 and Rv1198 strongly interact to form a heterodimeric complex under reducing conditions, which is characterized by a dissociation constant of 97 × 10 − 9 M and melting temperature, Tm, of 50.5 °C. Strong humoral responses to Rv1197, Rv1198, CFP-10 and MoaC1 (Rv3111) antigens were found in Indian patients with active pulmonary tuberculosis (n = 44), in comparison to non-infected healthy individuals (n = 20). The seroreactivity to Rv1198 was characterized by a sensitivity of 75% and specificity of 90%. In BALB/c mice, immunization with Rv1198-FIA induced a pro-inflammatory response with elevated levels of TNF and IL-6, along with low induction of IFN-γ, IL-2 and IL-10, but no induction of IL-4. Conclusion Rv1197 and Rv1198 form a stable complex, which is regulated by the redox state of Rv1198. Rv1198 is immunogenic with highly specific seroreactivity towards TB patients' serum. Rv1198 elicits a pro-inflammatory recall response in immunized mice. General Significance This study characterizes the interaction of Rv1197 and Rv1198, and establishes the immunogenic nature of Rv1198.

The inhibition of human topoisomerase IIα (Hu-TopoIIα), a major enzyme involved in maintaining DNA topology, repair, and chromosome condensation/decondensation results in loss of genomic integrity. In the present study, the inhibition of ATPase domain of Hu-TopoIIα as a possible mechanism of genotoxicity of 1,4-benzoquinone (BQ), hydroquinone (HQ), naphthoquinone (1,2-NQ and 1,4-NQ), and 9,10-phenanthroquinone (9,10-PQ) was investigated. In silico modeling predicted that 1,4-BQ, 1,2-NQ, 1,4-NQ, and 9,10-PQ could interact with Ser-148, Ser-149, Asn-150, and Asn-91 residues of the ATPase domain of Hu-TopoIIα. Biochemical inhibition assays with the purified ATPase domain of Hu-TopoIIα revealed that 1,4-BQ is the most potent inhibitor followed by 1,4-NQ > 1,2-NQ > 9,10-PQ > HQ. Ligand-binding studies using isothermal titration calorimetry revealed that 1,4-BQ, HQ, 1,4-NQ, 1,2-NQ, and 9,10-PQ enter into four sequentially binding site models inside the domain. 1,4-BQ exhibited the strongest binding, followed by 1,4-NQ > 1,2-NQ > 9,10-PQ > HQ, as revealed by their average K(d) values. The cellular fate of such inhibition was further evidenced by an increase in the number of Hu-TopoIIα-DNA cleavage complexes in the human lung epithelial cells (BEAS-2B) using trapped in agarose DNA immunostaining (TARDIS) assay, which utilizes antibody specific for Hu-TopoIIα. Furthermore, the increase in γ-H2A.X levels quantitated by flow cytometry and visualized by immunofluorescence microscopy illustrated that accumulation of DNA double-strand breaks inside the cells can be attributed to the inhibition of Hu-TopoIIα. These findings collectively suggest that 1,4-BQ, 1,2-NQ, 1,4-NQ, and 9,10-PQ inhibit the ATPase domain and potentially result in Hu-TopoIIα-mediated clastogenic and leukemogenic events.

Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) is induced in energy-starved conditions and is a key regulator of energy homeostasis. This makes PGC-1α an attractive therapeutic target for metabolic syndrome and diabetes. In our effort to identify new regulators of PGC-1α expression, we found that GW4064, a widely used synthetic agonist for the nuclear bile acid receptor [farnesoid X receptor (FXR)] strongly enhances PGC-1α promoter reporter activity, mRNA, and protein expression. This induction in PGC-1α concomitantly enhances mitochondrial mass and expression of several PGC-1α target genes involved in mitochondrial function. Using FXR-rich or FXR-nonexpressing cell lines and tissues, we found that this effect of GW4064 is not mediated directly by FXR but occurs via activation of estrogen receptor-related receptor α (ERRα). Cell-based, biochemical and biophysical assays indicate GW4064 as an agonist of ERR proteins. Interestingly, FXR disruption alters GW4064 induction of PGC-1α mRNA in a tissue-dependent manner. Using FXR-null [FXR knockout (FXRKO)] mice, we determined that GW4064 induction of PGC-1α expression is not affected in oxidative soleus muscles of FXRKO mice but is compromised in the FXRKO liver. Mechanistic studies to explain these differences revealed that FXR physically interacts with ERR and protects them from repression by the atypical corepressor, small heterodimer partner in liver. Together, this interplay between ERRα-FXR-PGC-1α and small heterodimer partner offers new insights into the biological functions of ERRα and FXR, thus providing a knowledge base for therapeutics in energy balance-related pathophysiology.

Rv3619c and Rv3620c are the secretory, antigenic proteins of the ESAT-6/CFP-10 family of Mycobacterium tuberculosis H37Rv. In this article, we show that Rv3619c interacts with Rv3620c to form a 1 : 1 heterodimeric complex with a dissociation constant (K(d)) of 4.8 × 10(-7) M. The thermal unfolding of the heterodimer was completely reversible, with a T(m) of 48 °C. The comparative thermodynamics and thermal unfolding analysis of the Rv3619c-Rv3620c dimer, the ESAT-6-CFP-10 dimer and another ESAT family heterodimer, Rv0287-Rv0288, revealed that the binding strength and stability of Rv3619c-Rv3620c are relatively lower than those of the other two pairs. Molecular modeling and docking studies predict the structure of Rv3619c-Rv3620c to be similar to that of ESAT-6-CFP-10. Spectroscopic studies revealed that, in an acidic environment, Rv3619c and Rv3620c lose their secondary structure and interact weakly to form a complex with a lower helical content, indicating that Rv3619c-Rv3620c is destabilized at low pH. These results, combined with those of previous studies, suggest that unfolding of the proteins is required for dissociation of the complex and membrane binding. In the presence of membrane mimetics, the α-helical contents of Rv3619c and Rv3620 increased by 42% and 35%, respectively. In mice, the immune response against Rv3619c protein is characterized by increased levels of interferon-γ, interleukin-12 and IgG(2a) , indicating a dominant Th1 response, which is mandatory for protection against mycobacterial infection. This study therefore emphasizes the potential of Rv3619c as a subunit vaccine candidate.

A rapid, sensitive and simple reverse-phase high-performance liquid chromatographic–electrospray ionization–mass spectrometry method for simultaneous determination of cleomiscosin A and cleomiscosin B has been developed and validated. The isomeric coumarinolignoids cleomiscosin A (1) and cleomiscosin B (2) were separated on a Waters symmetry C18 column with a solvent system composed of acetonitrile–methanol (1:2) and acetic acid–water (0.5 : 99.5) in a gradient elution mode. The absorption at 326 nm was chosen as the measuring wavelength in which resolution and baseline separation of compounds 1 and 2 could be obtained. The identity of the two isomeric compounds 1 and 2 in the samples were determined on a triple quadrupole mass spectrometer with ESI interface operating in the positive mode. Calibration curves were linear (r2 > 0.993) over the concentration range 20–200 µg/mL for cleomiscosin A and 10–200 µg/mL for cleomiscosin B with acceptable accuracy and precision, respectively. The intra-day and inter-day precision were 1.13 and 0.82% for cleomiscosin A and 1.78 and 1.28% for cleomiscosin B, respectively. The validated method was successfully applied for the analysis of the above two compounds in different extracts of Cleome viscosa. Copyright © 2008 John Wiley & Sons, Ltd.

A simple, accurate and reproducible reverse-phase HPLC method has been developed for identification and quantification of two isomeric coumarinolignoids, cleomiscosin A and B in different extracts of the seeds of Cleome viscosa using photodiode array detection at 326 nm. Cleomiscosin A and B were separated on a Waters symmetry C18 column (250 × 4.6 mm with 5.0 µm particle size) with an isocratic elution system composed of acetonitrile–methanol (1:2, v/v) and acetic acid–water (0.5:99.5, v/v) in the ratio of 40:60 (v/v). The calibration curves were linear (r2 > 0.997) in the concentration ranges of 20–100 µg/mL for both compounds. The limits of detection and quantification were 15 and 20 µg/mL for both cleomiscosin A and B. The intra- and inter-day precisions were 3.68 and 2.22% for cleomiscosin A and 4.22 and 5.06% for cleomiscosin B. The recoveries measured at two different concentration levels varied from 98.03 to 110.06%. The method was used to identify and quantify cleomiscosins A and B in different extracts of Cleome viscosa seeds. Copyright © 2007 John Wiley & Sons, Ltd.

Steganography conceals the very existence of the message by engrafting it inside a carrier file of some type. In this paper, payload image is distributed into three different images; Normalized Error (NE), Error Factor (EF) and Sign Map (SM) by using the primary cover image. These three images are then embedded in a secondary cover image by using an existing high capacity image steganography algorithm. The experimental results show that the Normalized error (NE) and Error factor (EF) are different from payload while Sign Map (SM) contains only binary values. Therefore, No one can derive the original payload using normalized factor, error factor and sign map until s/he has a primary cover.

Toxoplasma gondii ADF (TgADF) belongs to a functional subtype characterized by strong G-actin sequestering activity and low F-actin severing activity. Among the characterized ADF/cofilin proteins, TgADF has the shortest length and is missing a C-terminal helix implicated in F-actin binding. In order to understand its characteristic properties, we have determined the solution structure of TgADF and studied its backbone dynamics from 15N-relaxation measurements. TgADF has conserved ADF/cofilin fold consisting of a central mixed β-sheet comprised of six β-strands that are partially surrounded by three α-helices and a C-terminal helical turn. The high G-actin sequestering activity of TgADF relies on highly structurally and dynamically optimized interactions between G-actin and G-actin binding surface of TgADF. The equilibrium dissociation constant for TgADF and rabbit muscle G-actin was 23.81 nM, as measured by ITC, which reflects very strong affinity of TgADF and G-actin interactions. The F-actin binding site of TgADF is partially formed, with a shortened F-loop that does not project out of the ellipsoid structure and a C-terminal helical turn in place of the C-terminal helix α4. Yet, it is more rigid than the F-actin binding site of Leishmania donovani cofilin. Experimental observations and structural features do not support the interaction of PIP2 with TgADF, and PIP2 does not affect the interaction of TgADF with G-actin. Overall, this study suggests that conformational flexibility of G-actin binding sites enhances the affinity of TgADF for G-actin, while conformational rigidity of F-actin binding sites of conventional ADF/cofilins is necessary for stable binding to F-actin.