Rv1027c-Rv1028c encode functional KdpDE two - Component system in Mycobacterium tuberculosis

In Mycobacterium tuberculosis Rv1027c-Rv1028c genes are predicted to encode KdpDE two component system, which is highly conserved across all bacterial species. Here, we show that the system is functionally active and KdpD sensor kinase undergoes autophosphorylation and transfers phosphoryl group to KdpE, response regulator protein. We identified His(642) and Asp(52) as conserved phosphorylation sites in KdpD and KdpE respectively and by SPR analysis confirmed the physical interaction between them. KdpD was purified with prebound divalent ions and their importance in phosphorylation was established using protein refolding and ion chelation approaches. Genetically a single transcript encoded both KdpD and KdpE proteins. Overall, we report that M. tuberculosis KdpDE system operates like a canonical two component system. (C) 2014 Elsevier Inc. All rights reserved.

Tuning nuclearity of clusters by positional change of functional group: Synthesis of polynuclear clusters, crystal structures and magnetic properties

Four neutral polynuclear magnetic clusters, (Mn6Mn2Na2I)-Mn-III-Na-II(N-3)(8)(mu(1)-O)(2)(L-1)(6)(CH3OH)(2)] (1), (Mn6Na2I)-Na-III(N-3)(4)(mu(4)-O)(2)(L-2)(4)(CH3COO)(4)] (2), Ni-5(II)(N-3)(4)(HL1)(4)(HCOO)(2)(CH3OH)(2)(H2O)(2)]center dot 2CH(3)OH (3) and (Ni4Na2I)-Na-II(N-3)(4)(HL2)(6)]center dot 2CH(3)OH (4) have been synthesized using tetradentate ligands H2L1-2 along with azide as a co-ligand. H2L1-2 are the products formed in situ upon condensation of 2-hydroxy-3-methoxybenzaldehyde with 1-aminopropan-2-ol and 1-aminopropan-3-ol, respectively. Single crystal X-ray diffraction and bond valence sum calculation showed that complex 1 is composed of both Mn-III and Mn-II. Complex 3 contains coordinated formate, which was formed upon in situ oxidation of methanol. The magnetic study over a wide range of temperatures of all the complexes (1-4) showed that 1 and 2 are antiferromagnetic whereas other two (3-4) are predominantly ferromagnetic. The estimated ground states of the complexes are S approximate to 3(1), S = 4(2), S = 5(3) and S approximate to 4(4), respectively. (C) 2014 Elsevier B.V. All rights reserved.

A Genetic Analysis of the Functional Interactions within Mycobacterium tuberculosis Single-Stranded DNA Binding Protein

Single-stranded DNA binding proteins (SSBs) are vital in all organisms. SSBs of Escherichia coli (EcoSSB) and Mycobacterium tuberculosis (MtuSSB) are homotetrameric. The N-terminal domains (NTD) of these SSBs (responsible for their tetramerization and DNA binding) are structurally well defined. However, their C-terminal domains (CTD) possess undefined structures. EcoSSB NTD consists of beta 1-beta 1'-beta 2-beta 3-alpha-beta 4-beta 45(1)-beta 45(2)-beta 5 secondary structure elements. MtuSSB NTD includes an additional beta-strand (beta 6) forming a novel hook-like structure. Recently, we observed that MtuSSB complemented an E. coli Delta ssb strain. However, a chimeric SSB (m beta 4-beta 5), wherein only the terminal part of NTD (beta 4-beta 5 region possessing L-45 loop) of EcoSSB was substituted with that from MtuSSB, failed to function in E. coli in spite of its normal DNA binding and oligomerization properties. Here, we designed new chimeras by transplanting selected regions of MtuSSB into EcoSSB to understand the functional significance of the various secondary structure elements within SSB. All chimeric SSBs formed homotetramers and showed normal DNA binding. The m beta 4-beta 6 construct obtained by substitution of the region downstream of beta 5 in m beta 4-beta 5 SSB with the corresponding region (beta 6) of MtuSSB complemented the E. coli strain indicating a functional interaction between the L-45 loop and the beta 6 strand of MtuSSB.

Cu-II-Azide Polynuclear Complexes of Three Different Building Clusters with the Same Schiff-Base Ligand: Synthesis, Structures, Magnetic Behavior, and Density Functional Theory Studies

Three copper-azido complexes Cu-4(N-3)(8)(L-1)(2)(MeOH)(2)](n) (1), Cu-4(N-3)(8)(L-1)(2)] (2), and Cu-5(N-3)(10)(L-1)(2)](n) (3) L-1 is the imine resulting from the condensation of pyridine-2-carboxaldehyde with 2-(2-pyridyl)ethylamine] have been synthesized using lower molar equivalents of the Schiff base ligand with Cu(NO3)(2)center dot 3H(2)O and an excess of NaN3. Single crystal X-ray structures show that the basic unit of the complexes 1 and 2 contains Cu-4(II) building blocks; however, they have distinct basic and overall structures due to a small change in the bridging mode of the peripheral pair of copper atoms in the linear tetranudear structures. Interestingly, these changes are the result of changing the solvent system (MeOH/H2O to EtOH/H2O) used for the synthesis, without changing the proportions of the components (metal to ligand ratio 2:1). Using even lower proportions of the ligand, another unique complex was isolated with Cu-5(II) building units, forming a two-dimensional complex (3). Magnetic susceptibility measurements over a wide range of temperature exhibit the presence of both antiferromagnetic (very weak) and ferromagnetic exchanges within the tetranuclear unit structures. Density functional theory calculations (using B3LYP functional, and two different basis sets) have been performed on the complexes 1 and 2 to provide a qualitative theoretical interpretation of their overall magnetic behavior.

A FUNCTIONAL MODEL FOR PURE Gamma-CONTRACTIONS

A pair of commuting operators (S,P) defined on a Hilbert space H for which the closed symmetrized bidisc Gamma = {(z(1) + z(2), z(1)z(2)) : vertical bar z(1)vertical bar <= 1, vertical bar z(2)vertical bar <= 1} subset of C-2 is a spectral set is called a Gamma-contraction in the literature. A Gamma-contraction (S, P) is said to be pure if P is a pure contraction, i.e., P*(n) -> 0 strongly as n -> infinity Here we construct a functional model and produce a set of unitary invariants for a pure Gamma-contraction. The key ingredient in these constructions is an operator, which is the unique solution of the operator equation S - S*P = DpXDp, where X is an element of B(D-p), and is called the fundamental operator of the Gamma-contraction (S, P). We also discuss some important properties of the fundamental operator.

Smoothed Functional Algorithms for Stochastic Optimization Using q-Gaussian Distributions

Smoothed functional (SF) schemes for gradient estimation are known to be efficient in stochastic optimization algorithms, especially when the objective is to improve the performance of a stochastic system However, the performance of these methods depends on several parameters, such as the choice of a suitable smoothing kernel. Different kernels have been studied in the literature, which include Gaussian, Cauchy, and uniform distributions, among others. This article studies a new class of kernels based on the q-Gaussian distribution, which has gained popularity in statistical physics over the last decade. Though the importance of this family of distributions is attributed to its ability to generalize the Gaussian distribution, we observe that this class encompasses almost all existing smoothing kernels. This motivates us to study SF schemes for gradient estimation using the q-Gaussian distribution. Using the derived gradient estimates, we propose two-timescale algorithms for optimization of a stochastic objective function in a constrained setting with a projected gradient search approach. We prove the convergence of our algorithms to the set of stationary points of an associated ODE. We also demonstrate their performance numerically through simulations on a queuing model.

Newton-based stochastic optimization using q-Gaussian smoothed functional algorithms

We present the first q-Gaussian smoothed functional (SF) estimator of the Hessian and the first Newton-based stochastic optimization algorithm that estimates both the Hessian and the gradient of the objective function using q-Gaussian perturbations. Our algorithm requires only two system simulations (regardless of the parameter dimension) and estimates both the gradient and the Hessian at each update epoch using these. We also present a proof of convergence of the proposed algorithm. In a related recent work (Ghoshdastidar, Dukkipati, & Bhatnagar, 2014), we presented gradient SF algorithms based on the q-Gaussian perturbations. Our work extends prior work on SF algorithms by generalizing the class of perturbation distributions as most distributions reported in the literature for which SF algorithms are known to work turn out to be special cases of the q-Gaussian distribution. Besides studying the convergence properties of our algorithm analytically, we also show the results of numerical simulations on a model of a queuing network, that illustrate the significance of the proposed method. In particular, we observe that our algorithm performs better in most cases, over a wide range of q-values, in comparison to Newton SF algorithms with the Gaussian and Cauchy perturbations, as well as the gradient q-Gaussian SF algorithms. (C) 2014 Elsevier Ltd. All rights reserved.

Effect of functional groups on separating carbon dioxide from CO2/N-2 gas mixtures using edge functionalized graphene nanoribbons

Development of microporous adsorbents for separation and sequestration of carbon dioxide from flue gas streams is an area of active research. In this study, we assess the influence of specific functional groups on the adsorption selectivity of CO2/N-2 mixtures through Grand Canonical Monte Carlo (GCMC) simulations. Our model system consists of a bilayer graphene nanoribbon that has been edge functionalized with OH, NH2, NO2, CH3 and COOH. Ab initio Moller-Plesset (MP2) calculations with functionalized benzenes are used to obtain binding energies and optimized geometries for CO2 and N-2. This information is used to validate the choice classical forcefields in GCMC simulations. In addition to simulations of adsorption from binary mixtures of CO2 and N-2, the ideal adsorbed solution theory (IAST) is used to predict mixture isotherms. Our study reveals that functionalization always leads to an increase in the adsorption of both CO2 and N-2 with the highest for COOH. However, significant enhancement in the selectivity for CO2 is only seen with COOH functionalized nanoribbons. The COOH functionalization gives a 28% increase in selectivity compared to H terminated nanoribbons, whereas the improvement in the selectivity for other functional groups are much Enure modest. Our study suggests that specific functionalization with COOH groups can provide a material's design strategy to improve CO2 selectivity in microporous adsorbents. Synthesis of graphene nanoplatelets with edge functionalized COOH, which has the potential for large scale production, has recently been reported (Jeon el, al., 2012). (C) 2014 Elsevier Ltd. All rights reserved,

Direct regulation of topoisomerase activity by a nucleoid-associated protein

The topological homeostasis of bacterial chromosomes is maintained by the balance between compaction and the topological organization of genomes. Two classes of proteins play major roles in chromosome organization: the nucleoid-associated proteins (NAPs) and topoisomerases. The NAPs bind DNA to compact the chromosome, whereas topoisomerases catalytically remove or introduce supercoils into the genome. We demonstrate that HU, a major NAP of Mycobacterium tuberculosis specifically stimulates the DNA relaxation ability of mycobacterial topoisomerase I (TopoI) at lower concentrations but interferes at higher concentrations. A direct physical interaction between M. tuberculosis HU (MtHU) and TopoI is necessary for enhancing enzyme activity both in vitro and in vivo. The interaction is between the amino terminal domain of MtHU and the carboxyl terminal domain of TopoI. Binding of MtHU did not affect the two catalytic trans-esterification steps but enhanced the DNA strand passage, requisite for the completion of DNA relaxation, a new mechanism for the regulation of topoisomerase activity. An interaction-deficient mutant of MtHU was compromised in enhancing the strand passage activity. The species-specific physical and functional cooperation between MtHU and TopoI may be the key to achieve the DNA relaxation levels needed to maintain the optimal superhelical density of mycobacterial genomes.

Paralogous cAMP Receptor Proteins in Mycobacterium smegmatis Show Biochemical and Functional Divergence

The cyclic AMP receptor protein (CRP) family of transcription factors consists of global regulators of bacterial gene expression. Here, we identify two paralogous CRPs in the genome of Mycobacterium smegmatis that have 78% identical sequences and characterize them biochemically and functionally. The two proteins (MSMEG_0539 and MSMEG_6189) show differences in cAMP binding affinity, trypsin sensitivity, and binding to a CRP site that we have identified upstream of the msmeg_3781 gene. MSMEG_6189 binds to the CRP site readily in the absence of cAMP, while MSMEG_0539 binds in the presence of cAMP, albeit weakly. msmeg_6189 appears to be an essential gene, while the ?msmeg_0539 strain was readily obtained. Using promoter-reporter constructs, we show that msmeg_3781 is regulated by CRP binding, and its transcription is repressed by MSMEG_6189. Our results are the first to characterize two paralogous and functional CRPs in a single bacterial genome. This gene duplication event has subsequently led to the evolution of two proteins whose biochemical differences translate to differential gene regulation, thus catering to the specific needs of the organism.

Chemical specificity and conformational flexibility in proteinase-inhibitor interaction: Scaffolds for promiscuous binding

One of the most important roles of proteins in cellular milieu is recognition of other biomolecules including other proteins. Protein protein complexes are involved in many essential cellular processes. Interfaces of protein protein complexes are traditionally known to be conserved in evolution and less flexible than other solvent interacting tertiary structural surface. But many examples are emerging where these features do not hold good. An understanding of inter-play between flexibility and sequence conservation is emerging, providing a fresh dimension to the paradigm of sequence structure function relationship. The functional manifestation of the inter-relation between sequence conservation and flexibility of interface is exemplified in this review using proteinase inhibitor protein complexes. (C) 2014 Elsevier Ltd. All rights reserved.

Temporally distinct roles of ATM and ROS in genotoxic- stress-dependent induction and maintenance of cellular senescence

Cells exposed to genotoxic stress induce cellular senescence through a DNA damage response (DDR) pathway regulated by ATM kinase and reactive oxygen species (ROS). Here, we show that the regulatory roles for ATM kinase and ROS differ during induction and maintenance of cellular senescence. Cells treated with different genotoxic agents were analyzed using specific pathway markers and inhibitors to determine that ATM kinase activation is directly proportional to the dose of the genotoxic stress and that senescence initiation is not dependent on ROS or the p53 status of cells. Cells in which ROS was quenched still activated ATM and initiated the DDR when insulted, and progressed normally to senescence. By contrast, maintenance of a viable senescent state required the presence of ROS as well as activated ATM. Inhibition or removal of either of the components caused cell death in senescent cells, through a deregulated ATM-ROS axis. Overall, our work demonstrates existence of an intricate temporal hierarchy between genotoxic stress, DDR and ROS in cellular senescence. Our model reports the existence of different stages of cellular senescence with distinct regulatory networks.

Dendritic atrophy constricts functional maps in resonance and impedance properties of hippocampal model neurons

A gradient in the density of hyperpolarization-activated cyclic-nucleotide gated (HCN) channels is necessary for the emergence of several functional maps within hippocampal pyramidal neurons. Here, we systematically analyzed the impact of dendritic atrophy on nine such functional maps, related to input resistance and local/transfer impedance properties, using conductance-based models of hippocampal pyramidal neurons. We introduced progressive dendritic atrophy in a CA1 pyramidal neuron reconstruction through a pruning algorithm, measured all functional maps in each pruned reconstruction, and arrived at functional forms for the dependence of underlying measurements on dendritic length. We found that, across frequencies, atrophied neurons responded with higher efficiency to incoming inputs, and the transfer of signals across the dendritic tree was more effective in an atrophied reconstruction. Importantly, despite the presence of identical HCN-channel density gradients, spatial gradients in input resistance, local/transfer resonance frequencies and impedance profiles were significantly constricted in reconstructions with dendrite atrophy, where these physiological measurements across dendritic locations converged to similar values. These results revealed that, in atrophied dendritic structures, the presence of an ion channel density gradient alone was insufficient to sustain homologous functional maps along the same neuronal topograph. We assessed the biophysical basis for these conclusions and found that this atrophy-induced constriction of functional maps was mediated by an enhanced spatial spread of the influence of an HCN-channel cluster in atrophied trees. These results demonstrated that the influence fields of ion channel conductances need to be localized for channel gradients to express themselves as homologous functional maps, suggesting that ion channel gradients are necessary but not sufficient for the emergence of functional maps within single neurons.

Study of structures, energies and vibrational frequencies of (O-2)(n)(+) (n=2-5) clusters by GGA and meta-GGA density functional methods

Using Generalized Gradient Approximation (GGA) and meta-GGA density functional methods, structures, binding energies and harmonic vibrational frequencies for the clusters O-4(+), O-6(+), O-8(+) and O-10(+) have been calculated. The stable structures of O-4(+), O-6(+), O-8(+) and O-10(+) have point groups D-2h, D-3h, D-4h, and D-5h optimized on the quartet, sextet, octet and dectet potential energy surfaces, respectively. Rectangular (D-2h) O-4(+) has been found to be more stable compared to trans-planar (C-2h) on the quartet potential energy surface. Cyclic structure (D-3h) of CA cluster ion has been calculated to be more stable than other structures. Binding energy (B.E.) of the cyclic O-6(+) is in good agreement with experimental measurement. The zero-point corrected B.E. of O-8(+) with D4h symmetry on the octet potential energy surface and zero-point corrected B.E. of O-10(+) with D-5h symmetry on the dectet potential energy surface are also in good agreement with experimental values. The B.E. value for O-4(+) is close to the experimental value when single point energy is calculated by Brueckner coupled-cluster method, BD(T). (C) 2014 Elsevier B.V. All rights reserved.

Molecular and Functional Characterization of RecD, a Novel Member of the SF1 Family of Helicases, from Mycobacterium tuberculosis

Background: The heterotrimeric M. tuberculosis RecBCD complex, or each of its individual subunits, remains uncharacterized. Results: MtRecD exists as a homodimer in solution, catalyzes ssDNA-dependent ATP hydrolysis, unwinding of DNA replication/recombination intermediates, and interacts with RecA. Conclusion: MtRecD possesses strong 5 3- and weak 3 5-helicase activities. Significance: These findings provide insights into the mechanism underlying DSB repair and homologous recombination in mycobacteria. The annotated whole-genome sequence of Mycobacterium tuberculosis revealed the presence of a putative recD gene; however, the biochemical characteristics of its encoded protein product (MtRecD) remain largely unknown. Here, we show that MtRecD exists in solution as a stable homodimer. Protein-DNA binding assays revealed that MtRecD binds efficiently to single-stranded DNA and linear duplexes containing 5 overhangs relative to the 3 overhangs but not to blunt-ended duplex. Furthermore, MtRecD bound more robustly to a variety of Y-shaped DNA structures having 18-nucleotide overhangs but not to a similar substrate containing 5-nucleotide overhangs. MtRecD formed more salt-tolerant complexes with Y-shaped structures compared with linear duplex having 3 overhangs. The intrinsic ATPase activity of MtRecD was stimulated by single-stranded DNA. Site-specific mutagenesis of Lys-179 in motif I abolished the ATPase activity of MtRecD. Interestingly, although MtRecD-catalyzed unwinding showed a markedly higher preference for duplex substrates with 5 overhangs, it could also catalyze significant unwinding of substrates containing 3 overhangs. These results support the notion that MtRecD is a bipolar helicase with strong 5 3 and weak 3 5 unwinding activities. The extent of unwinding of Y-shaped DNA structures was approximate to 3-fold lower compared with duplexes with 5 overhangs. Notably, direct interaction between MtRecD and its cognate RecA led to inhibition of DNA strand exchange promoted by RecA. Altogether, these studies provide the first detailed characterization of MtRecD and present important insights into the type of DNA structure the enzyme is likely to act upon during the processes of DNA repair or homologous recombination.