Functional Analysis of DNA Replication Fork Reversal Catalyzed by Mycobacterium tuberculosis RuvAB Proteins

Initially discovered in Escherichia coli, RuvAB proteins are ubiquitous in bacteria and play a dual role as molecular motor proteins responsible for branch migration of the Holliday junction(s) and reversal of stalled replication forks. Despite mounting genetic evidence for a crucial role of RuvA and RuvB proteins in reversal of stalled replication forks, the mechanistic aspects of this process are still not fully understood. Here, we elucidate the ability of Mycobacterium tuberculosis RuvAB (MtRuvAB) complex to catalyze the reversal of replication forks using a range of DNA replication fork substrates. Our studies show that MtRuvAB, unlike E. coli RuvAB, is able to drive replication fork reversal via the formation of Holliday junction intermediates, suggesting that RuvAB-catalyzed fork reversal involves concerted unwinding and annealing of nascent leading and lagging strands. We also demonstrate the reversal of replication forks carrying hemi-replicated DNA, indicating that MtRuvAB complex-catalyzed fork reversal is independent of symmetry at the fork junction. The fork reversal reaction catalyzed by MtRuvAB is coupled to ATP hydrolysis, is processive, and culminates in the formation of an extended reverse DNA arm. Notably, we found that sequence heterology failed to impede the fork reversal activity of MtRuvAB. We discuss the implications of these results in the context of recognition and processing of varied types of replication fork structures by RuvAB proteins.

Solution of a class of surface water wave scattering problems involving non-reflecting vertical barriers

A large class of scattering problems of surface water waves by vertical barriers lead to mixed boundary value problems for Laplace equation. Specific attentions are paid, in the present article, to highlight an analytical method to handle this class of problems of surface water wave scattering, when the barriers in question are non-reflecting in nature. A new set of boundary conditions is proposed for such non-reflecting barriers and tile resulting boundary value problems are handled in the linearized theory of water waves. Three basic poblems of scattering by vertical barriers are solved. The present new theory of non-reflecting vertical barriers predict new transmission coefficients and tile solutions of tile mathematical problems turn out to be extremely simple and straight forward as compared to the solution for other types of barriers handled previously.

Dimethyl sulfoxide induced structural transformations and non-monotonic concentration dependence of conformational fluctuation around active site of lysozyme

Experimental studies have observed significant changes in both structure and function of lysozyme (and other proteins) on addition of a small amount of dimethyl sulfoxide (DMSO) in aqueous solution. Our atomistic molecular dynamic simulations of lysozyme in water-DMSO reveal the following sequence of changes on increasing DMSO concentration. (i) At the initial stage (around 5% DMSO concentration) protein's conformational flexibility gets markedly suppressed. From study of radial distribution functions, we attribute this to the preferential solvation of exposed protein hydrophobic residues by the methyl groups of DMSO. (ii) In the next stage (10-15% DMSO concentration range), lysozome partially unfolds accompanied by an increase both in fluctuation and in exposed protein surface area. (iii) Between 15-20% concentration ranges, both conformational fluctuation and solvent accessible protein surface area suddenly decrease again indicating the formation of an intermediate collapse state. These results are in good agreement with near-UV circular dichroism (CD) and fluorescence studies. We explain this apparently surprising behavior in terms of a structural transformation which involves clustering among the methyl groups of DMSO. (iv) Beyond 20% concentration of DMSO, the protein starts its final sojourn towards the unfolding state with further increase in conformational fluctuation and loss in native contacts. Most importantly, analysis of contact map and fluctuation near the active site reveal that both partial unfolding and conformational fluctuations are centered mostly on the hydrophobic core of active site of lysozyme. Our results could offer a general explanation and universal picture of the anomalous behavior of protein structure-function observed in the presence of cosolvents (DMSO, ethanol, tertiary butyl alcohol, dioxane) at their low concentrations. (C) 2012 American Institute of Physics. [http://dx.doi.org/10.1063/1.3694268]

Comparison of tertiary structures of proteins in protein-protein complexes with unbound forms suggests prevalence of allostery in signalling proteins

Abstract: Background: Most signalling and regulatory proteins participate in transient protein-protein interactions during biological processes. They usually serve as key regulators of various cellular processes and are often stable in both protein-bound and unbound forms. Availability of high-resolution structures of their unbound and bound forms provides an opportunity to understand the molecular mechanisms involved. In this work, we have addressed the question "What is the nature, extent, location and functional significance of structural changes which are associated with formation of protein-protein complexes?" Results: A database of 76 non-redundant sets of high resolution 3-D structures of protein-protein complexes, representing diverse functions, and corresponding unbound forms, has been used in this analysis. Structural changes associated with protein-protein complexation have been investigated using structural measures and Protein Blocks description. Our study highlights that significant structural rearrangement occurs on binding at the interface as well as at regions away from the interface to form a highly specific, stable and functional complex. Notably, predominantly unaltered interfaces interact mainly with interfaces undergoing substantial structural alterations, revealing the presence of at least one structural regulatory component in every complex. Interestingly, about one-half of the number of complexes, comprising largely of signalling proteins, show substantial localized structural change at surfaces away from the interface. Normal mode analysis and available information on functions on some of these complexes suggests that many of these changes are allosteric. This change is largely manifest in the proteins whose interfaces are altered upon binding, implicating structural change as the possible trigger of allosteric effect. Although large-scale studies of allostery induced by small-molecule effectors are available in literature, this is, to our knowledge, the first study indicating the prevalence of allostery induced by protein effectors. Conclusions: The enrichment of allosteric sites in signalling proteins, whose mutations commonly lead to diseases such as cancer, provides support for the usage of allosteric modulators in combating these diseases.

Rationalization and prediction of drug resistant mutations in targets for clinical anti-tubercular drugs

Resistance to therapy limits the effectiveness of drug treatment in many diseases. Drug resistance can be considered as a successful outcome of the bacterial struggle to survive in the hostile environment of a drug-exposed cell. An important mechanism by which bacteria acquire drug resistance is through mutations in the drug target. Drug resistant strains (multi-drug resistant and extensively drug resistant) of Mycobacterium tuberculosis are being identified at alarming rates, increasing the global burden of tuberculosis. An understanding of the nature of mutations in different drug targets and how they achieve resistance is therefore important. An objective of this study is to first decipher sequence as well as structural bases for the observed resistance in known drug resistant mutants and then to predict positions in each target that are more prone to acquiring drug resistant mutations. A curated database containing hundreds of mutations in the 38 drug targets of nine major clinical drugs, associated with resistance is studied here. Mutations have been classified into those that occur in the binding site itself, those that occur in residues interacting with the binding site and those that occur in outer zones. Structural models of the wild type and mutant forms of the target proteins have been analysed to seek explanations for reduction in drug binding. Stability analysis of an entire array of 19 mutations at each of the residues for each target has been computed using structural models. Conservation indices of individual residues, binding sites and whole proteins are computed based on sequence conservation analysis of the target proteins. The analyses lead to insights about which positions in the polypeptide chain have a higher propensity to acquire drug resistant mutations. Thus critical insights can be obtained about the effect of mutations on drug binding, in terms of which amino acid positions and therefore which interactions should not be heavily relied upon, which in turn can be translated into guidelines for modifying the existing drugs as well as for designing new drugs. The methodology can serve as a general framework to study drug resistant mutants in other micro-organisms as well.

Catalytic activity of Peptidase N is required for adaptation of Escherichia coli to nutritional downshift and high temperature stress

Peptidase N (PepN), the sole M1 family member in Escherichia coli, displays broad substrate specificity and modulates stress responses: it lowers resistance to sodium salicylate (NaSal)-induced stress but is required during nutritional downshift and high temperature (NDHT) stress. The expression of PepN does not significantly change during different growth phases in LB or NaSal-induced stress; however, PepN amounts are lower during NDHT stress. To gain mechanistic insights on the roles of catalytic activity of PepN in modulating these two stress responses, alanine mutants of PepN replacing E264 (GAMEN motif) and E298 (HEXXH motif) were generated. There are no major structural changes between purified wild type (WT) and mutant proteins, which are catalytically inactive. Importantly, growth profiles of Delta pepN upon expression of WT or mutant proteins demonstrated the importance of catalytic activity during NDHT but not NaSal-induced stress. Further fluorescamine reactivity studies demonstrated that the catalytic activity of PepN is required to generate higher intracellular amounts of free N-terminal amino acids; consequently, the lower growth of Delta pepN during NDHT stress increases with high amounts of casamino acids. Together, this study sheds insights on the expression and functional roles of the catalytic activity of PepN during adaptation to NDHT stress. (C) 2012 Elsevier GmbH. All rights reserved.

Effect of signal peptide on stability and folding of escherichia coli thioredoxin

The signal peptide plays a key role in targeting and membrane insertion of secretory and membrane proteins in both prokaryotes and eukaryotes. In E. coli, recombinant proteins can be targeted to the periplasmic space by fusing naturally occurring signal sequences to their N-terminus. The model protein thioredoxin was fused at its N-terminus with malE and pelB signal sequences. While WT and the pelB fusion are soluble when expressed, the malE fusion was targeted to inclusion bodies and was refolded in vitro to yield a monomeric product with identical secondary structure to WT thioredoxin. The purified recombinant proteins were studied with respect to their thermodynamic stability, aggregation propensity and activity, and compared with wild type thioredoxin, without a signal sequence. The presence of signal sequences leads to thermodynamic destabilization, reduces the activity and increases the aggregation propensity, with malE having much larger effects than pelB. These studies show that besides acting as address labels, signal sequences can modulate protein stability and aggregation in a sequence dependent manner.

How much land-based greenhouse gas mitigation can be achieved without compromising food security and environmental goals?

Feeding 9-10billion people by 2050 and preventing dangerous climate change are two of the greatest challenges facing humanity. Both challenges must be met while reducing the impact of land management on ecosystem services that deliver vital goods and services, and support human health and well-being. Few studies to date have considered the interactions between these challenges. In this study we briefly outline the challenges, review the supply- and demand-side climate mitigation potential available in the Agriculture, Forestry and Other Land Use AFOLU sector and options for delivering food security. We briefly outline some of the synergies and trade-offs afforded by mitigation practices, before presenting an assessment of the mitigation potential possible in the AFOLU sector under possible future scenarios in which demand-side measures codeliver to aid food security. We conclude that while supply-side mitigation measures, such as changes in land management, might either enhance or negatively impact food security, demand-side mitigation measures, such as reduced waste or demand for livestock products, should benefit both food security and greenhouse gas (GHG) mitigation. Demand-side measures offer a greater potential (1.5-15.6Gt CO2-eq. yr(-1)) in meeting both challenges than do supply-side measures (1.5-4.3Gt CO2-eq. yr(-1) at carbon prices between 20 and 100US$ tCO(2)-eq. yr(-1)), but given the enormity of challenges, all options need to be considered. Supply-side measures should be implemented immediately, focussing on those that allow the production of more agricultural product per unit of input. For demand-side measures, given the difficulties in their implementation and lag in their effectiveness, policy should be introduced quickly, and should aim to codeliver to other policy agenda, such as improving environmental quality or improving dietary health. These problems facing humanity in the 21st Century are extremely challenging, and policy that addresses multiple objectives is required now more than ever.

Catalytic performance of highly dispersed Ni/TiO2 for dry and steam reforming of methane

The present study reports a sonochemical-assisted synthesis of a highly active and coke resistant Ni/TiO2 catalyst for dry and steam reforming of methane. The catalyst was characterized using XRD, TEM, XPS, BET analyzer and TGA/DTA techniques. The TEM analysis showed that Ni nanoparticles were uniformly dispersed on TiO2 surface with a narrow size distribution. The catalyst prepared via this approach exhibited excellent activity and stability for both the reactions compared to the reference catalyst prepared from the conventional wet impregnation method. For dry reforming, 86% CH4 conversion and 84% CO2 conversion was obtained at 700 degrees C. Nearly 92% CH4 conversion and 77% CO selectivity was observed under a H2O/CH4 ratio of 1.2 at 700 degrees C for the steam reforming reaction. In particular, the present catalyst is extremely active and resistant to coke formation for steam reforming at low steam/carbon ratios. There is no significant modification of Ni particles size and no coke deposition, even after a long term reaction, demonstrating its potential applicability as an industrial reformate for hydrogen production. The detailed kinetic studies have been presented for steam reforming and the mechanism involving Langmuir-Hinshelwood kinetics with adsorptive dissociation of CH4 as a rate determining step has been used to correlate the experimental data.

Kinematic and Mechanical Profile of the Self-Actuation of Thermosalient Crystal Twins of 1,2,4,5-Tetrabromobenzene: A Molecular Crystalline Analogue of a Bimetallic Strip

A paradigm shift from hard to flexible, organic-based optoelectronics requires fast and reversible mechanical response from actuating materials that are used for conversion of heat or light into mechanical motion. As the limits in the response times of polymer-based actuating materials are reached, which are inherent to the less-than-optimal coupling between the light/heat and mechanical energy in them, 1 a conceptually new approach to mechanical actuation is required to leapfrog the performance of organic actuators. Herein, we explore single crystals of 1,2,4,5-tetrabromobenzene (TBB) as actuating elements and establish relations between their kinematic profile and mechanical properties. Centimeter-size acicular crystals of TBB are the only naturally twinned crystals out of about a dozen known materials that exhibit the thermosalient effect-an extremely rare and visually impressive crystal locomotion. When taken over a phase transition, crystals of this material store mechanical strain and are rapidly self-actuated to sudden jumps to release the internal strain, leaping up to several centimeters. To establish the structural basis for this colossal crystal motility, we investigated the mechanical profile of the crystals from macroscale, in response to externally induced deformation under microscope, to nanoscale, by using nanoindentation. Kinematic analysis based on high-speed recordings of over 200 twinned TBB crystals exposed to directional or nondirectional heating unraveled that the crystal locomotion is a kinematically complex phenomenon that includes at least six kinematic effects. The nanoscale tests confirm the highly elastic nature, with an elastic deformation recovery (60%) that is far superior to those of molecular crystals reported earlier. This property appears to be critical for accumulation of stress required for crystal jumping. Twinned crystals of TBB exposed to moderate directional heating behave as all-organic analogue of a bimetallic `strip, where the lattice misfit between the two crystal components drives reveriible deformation of the crystal.

Plasmodium berghei glycine cleavage system T-protein is non-essential for parasite survival in vertebrate and invertebrate hosts

T-protein, an aminomethyltransferase, represents one of the four components of glycine cleavage system (GCS) and catalyzes the transfer of methylene group from H-protein intermediate to tetrahydrofolate (THF) forming N-5, N-10-methylene THF (CH2-THF) with the release of ammonia. The malaria parasite genome encodes T-, H- and L-proteins, but not P-protein which is a glycine decarboxylase generating the aminomethylene group. A putative GCS has been considered to be functional in the parasite mitochondrion despite the absence of a detectable P-protein homologue. In the present study, the mitochondrial localization of T-protein in the malaria parasite was confirmed by immunofluorescence and its essentiality in the entire parasite life cycle was studied by targeting the T-protein locus in Plasmodium berghei (Pb). PbT knock out parasites did not show any growth defect in asexual, sexual and liver stages indicating that the T-protein is dispensable for parasite survival in vertebrate and invertebrate hosts. The absence of P-protein homologue and the non-essentiality of T protein suggest the possible redundancy of GCS activity in the malaria parasite. Nevertheless, the H- and L-proteins of GCS could be essential for malaria parasite because of their involvement in alpha-lcetoacid dehydrogenase reactions. (C) 2014 Elsevier B.V. All rights reserved.

HuR Displaces Polypyrimidine Tract Binding Protein To Facilitate La Binding to the 3 ` Untranslated Region and Enhances Hepatitis C Virus Replication

HuR is a ubiquitous, RNA binding protein that influences the stability and translation of several cellular mRNAs. Here, we report a novel role for HuR, as a regulator of proteins assembling at the 3' untranslated region (UTR) of viral RNA in the context of hepatitis C virus (HCV) infection. HuR relocalizes from the nucleus to the cytoplasm upon HCV infection, interacts with the viral polymerase (NS5B), and gets redistributed into compartments of viral RNA synthesis. Depletion in HuR levels leads to a significant reduction in viral RNA synthesis. We further demonstrate that the interaction of HuR with the 3' UTR of the viral RNA affects the interaction of two host proteins, La and polypyrimidine tract binding protein (PTB), at this site. HuR interacts with La and facilitates La binding to the 3' UTR, enhancing La-mediated circularization of the HCV genome and thus viral replication. In addition, it competes with PTB for association with the 3' UTR, which might stimulate viral replication. Results suggest that HuR influences the formation of a cellular/viral ribonucleoprotein complex, which is important for efficient initiation of viral RNA replication. Our study unravels a novel strategy of regulation of HCV replication through an interplay of host and viral proteins, orchestrated by HuR. IMPORTANCE Hepatitis C virus (HCV) is highly dependent on various host factors for efficient replication of the viral RNA. Here, we have shown how a host factor (HuR) migrates from the nucleus to the cytoplasm and gets recruited in the protein complex assembling at the 3' untranslated region (UTR) of HCV RNA. At the 3' UTR, it facilitates circularization of the viral genome through interaction with another host factor, La, which is critical for replication. Also, it competes with the host protein PTB, which is a negative regulator of viral replication. Results demonstrate a unique strategy of regulation of HCV replication by a host protein through alteration of its subcellular localization and interacting partners. The study has advanced our knowledge of the molecular mechanism of HCV replication and unraveled the complex interplay between the host factors and viral RNA that could be targeted for therapeutic interventions.

Comparative proteomic analysis of primordial follicles from ovaries of immature and aged rats

Age related decline in reproductive performance in women is well documented and apoptosis has been considered as one of the reasons for the decline of primordial follicle reserve. Recently we observed a decline in the efficiency of DNA repair ability in aged rat primordial follicles as demonstrated by decreased mRNA levels of DNA repair genes BRCA1 and H2AX. In the present study, a two-dimensional electrophoresis (2DE) proteomic approach was employed to identify differentially expressed proteins in primordial follicles isolated from ovaries of immature (approximate to 20 days) and aged (approximate to 400-450 days) rats. Using MALDI-TOF/TOF MS, we identified 13 differentially expressed proteins (p<0.05) which included seven up-regulated and six down-regulated proteins in aged primordial follicles. These proteins are involved in a wide range of biological functions including apoptosis, DNA repair, and the immune system. Interestingly, the differentially expressed proteins such as FIGNL1 (DNA repair) and BOK (apoptotic protein) have not been previously reported in the rat primordial follicles and these proteins can be related to some common features of ovarian aging such as loss of follicle reserve and genome integrity. The quantitative differences of two important proteins BOK and FIGNL1 observed by the proteomic analysis were correlated with the transcript levels, as determined by semi-quantitative RT-PCR. Our results improve the current knowledge about protein factors associated with molecular changes in rat primordial follicles as a function of aging and our understanding of the proteomic processes involved in degenerative changes observed in aging primordial follicles.

NDK Interacts with FtsZ and Converts GDP to GTP to Trigger FtsZ Polymerisation - A Novel Role for NDK

Introduction Nucleoside diphosphate kinase (NDK), conserved across bacteria to humans, synthesises NTP from NDP and ATP. The eukaryotic homologue, the NDPK, uses ATP to phosphorylate the tubulin-bound GDP to GTP for tubulin polymerisation. The bacterial cytokinetic protein FtsZ, which is the tubulin homologue, also uses GTP for polymerisation. Therefore, we examined whether NDK can interact with FtsZ to convert FtsZ-bound GDP and/or free GDP to GTP to trigger FtsZ polymerisation. Methods Recombinant and native NDK and FtsZ proteins of Mycobacterium smegmatis and Mycobacterium tuberculosis were used as the experimental samples. FtsZ polymersation was monitored using 90 degrees light scattering and FtsZ polymer pelleting assays. The gamma 32P-GTP synthesised by NDK from GDP and gamma 32P-ATP was detected using thin layer chromatography and quantitated using phosphorimager. The FtsZ bound P-32-GTP was quantitated using phosphorimager, after UV-crosslinking, followed by SDS-PAGE. The NDK-FtsZ interaction was determined using Ni2+-NTA-pulldown assay and co-immunoprecipitation of the recombinant and native proteins in vitro and ex vivo, respectively. Results NDK triggered instantaneous polymerisation of GDP-precharged recombinant FtsZ in the presence of ATP, similar to the polymerisation of recombinant FtsZ (not GDP-precharged) upon the direct addition of GTP. Similarly, NDK triggered polymerisation of recombinant FtsZ (not GDP-precharged) in the presence of free GDP and ATP as well. Mutant NDK, partially deficient in GTP synthesis from ATP and GDP, triggered low level of polymerisation of MsFtsZ, but not of MtFtsZ. As characteristic of NDK's NTP substrate non-specificity, it used CTP, TTP, and UTP also to convert GDP to GTP, to trigger FtsZ polymerisation. The NDK of one mycobacterial species could trigger the polymerisation of the FtsZ of another mycobacterial species. Both the recombinant and the native NDK and FtsZ showed interaction with each other in vitro and ex vivo, alluding to the possibility of direct phosphorylation of FtsZ-bound GDP by NDK. Conclusion Irrespective of the bacterial species, NDK interacts with FtsZ in vitro and ex vivo and, through the synthesis of GTP from FtsZ-bound GDP and/or free GDP, and ATP (CTP/TTP/UTP), triggers FtsZ polymerisation. The possible biological context of this novel activity of NDK is presented.

Formation, microstructure and development of the localized shear deformation in low-carbon steels

An investigation has been made into the effect of microstructural parameters on the propensity for forming shear localization produced during high speed torsional testing by split Hopkinson bar with different average rates of 610, 650 and 1500 s(-1) in low carbon steels. These steels received the quenched, quenched and tempered as well as normalized treatments that provide wide microstructural parameters and mechanical properties. The results indicate that the occurrence of the shear localization is susceptible to the strength of the steels. In other words, the tendency of the quenched steel to form a shear band is higher than that of the other two steels. It is also found that there is a critical strain at which the shear localization occurs in the steels. The critical strain value is strongly dependent on the strength of the steels. Before arriving at this point, the material undergoes a slow work-hardening. After this point, the material suffers work-softening, corresponding to a process during which the deformation is gradually localized and eventually becomes spatially correlated to form a macroscopic shear band. Examinations by SEM reveal that the shear localization within the band involves a series of sequential crystallographic and non-crystallographic events including the change in crystal orientation, misorientation, generation and even perhaps damage in microstructures such as the initiation, growth and coalescence of the microcracks. It is expected that the sharp drop in the load-carrying capacity is associated with the growth and coalescence of the microcracks rather than the occurrence of the shear localization, but the shear localization is seen to accelerate the growth and coalescence of the microcracks. The thin foil observations by TEM reveal that the density of dislocations in the band is extremely high and the tangled arrangement and cell structure of dislocations tends to align along the shear direction. The multiplication and interaction of dislocations seems to be responsible for work-hardening of the steels. The avalanche of the dislocation cells corresponds to the sharp drop in shear stress at which the deformed specimen is broken. Double shear bands and kink bands are also observed in the present study. The principal band develops first and its width is narrower than that of the secondary band.