184 Small molecule identification against novel MDRA protein of Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) is an obstinate pathogen causing tuberculosis (TB) in Homo sapiens. One third of the World population is affected by Mtb (James et al., 2008). The multidrug-resistant protein-A (MDRA) belongs to ABC transporter family. The protein MDRA and the membrane integral protein MDRB together form the efflux pump (MDRA₂B₂ complex) that confers resistance by transport of the drugs out of the cell. The MDRB protein expression depends on the expression of MDRA (Baisakhee et al., 2002). In the present study, MDRA 3-D model (Figure) was generated with the help of comparative homology modeling techniques using pair-wise sequence alignment. The predicted 3-D model was subjected to refinement and validated. The active site of the protein was predicted. The virtual screening (VS) studies were performed at MDRB binding site with an in-house library of small molecules to identify a lead molecule that can inhibits the MDRA protein. The results of VS project competitive inhibitors of MDRB, for its binding with MDRA, and its drug-resistant activity. Hence, the MDRA protein may be treated as a novel target for the development of new chemical entities for tuberculosis therapy (Bhargavi et al., 2010; Malkhed et al., 2011).     

210 Computational study of substrates and mediators features of lacasses

Laccases are enzymes of the family multicopper oxidases, being widely used for biotechnological applications (Canas & Camarero, 2010). The enzymes’ catalytic cycle consists of the oxidation of the substrate with the concomitant reduction of molecular oxygen to water. In this process, the substrate is converted to a free radical, that can oxidize larger substrates acting as a mediator or it can undergo polymerization. Substrate binding is not specific, and there is a large diversity of substrates for laccases. Moreover, the binding site shows important differences among diverse species. The goal of the present work is to characterize the laccase binding pocket of different species, in order to establish their common pharmacophoric characteristics. For this purpose, we have carried out docking studies with a subset of substrates, covering the diversity of substrates using the Glide program (Friesner et al., 2004). We have also analyzed the characteristics of the binding site using diverse probes. We further have rationalized the differential values of km found among diverse species for a specific substrate. Finally, special attention has been devoted to the binding of the mediator 2,2′-azido-di-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS), commonly used in industrial processes. Figure 1 shows, ABTS docked onto the fungal laccase, whereas Figure 2 shows ABTS docked onto the bacterial laccase. The analysis of the protein–ligand complex together with the corresponding optimized geometries of the possible substrate species carried out using DFT suggest that the bound species is the protonated form of ABTS as previously suggested (Enguita et al., 2004). Furthermore, the results of this study also suggest that its mechanism of oxidation occurs in a similar way to the rest of substrates/mediators, in contrast to previous suggestions (Fabbrini et al., 2002).      

161 Discovery of potent KdsA inhibitors of Leptospira interrogans through homology modeling,docking, and molecular dynamics simulations

Leptospira interrogans is the foremost cause of human leptospirosis. Discovery of novel lead molecules for common drug targets of more than 250 Leptospira serovars is of significant research interest. Lipopolysaccharide (LPS) layer prevent entry of hydrophobic agents into the cell and protect structural integrity of the bacterium. KDO-8-phosphate synthase (KdsA) catalyzes the first step of KDO biosynthesis that leads to formation of inner core of LPS. KdsA was identified as a potential drug target against Leptospira interrogans through subtractive genomic approach, metabolic pathway analysis, and comparative analysis (Amineni et al., 2010). The present study rationalizes a systematic implementation of homology modeling, docking, and molecular dynamics simulations to discover potent KdsA inhibitors (Pradhan et al., 2013; Umamaheswari et al., 2010). A reliable tertiary structure of KdsA in complex with substrate PEP was constructed based on co-crystal structure of Aquifex aeolicus KdsA synthase with PEP using Modeller9v10. Geometry-based analog search for PEP was performed from LigandInfo database to generate an in house library of 352 ligands. The ligand data-set was docked into KdsA active site through three-stage docking technique (HTVS, SP, and XP) using Glidev5.7. Thirteen lead molecules were found to have better binding affinity compared to PEP (XP Gscore?=??7.38?kcal/mol; Figure 1). The best lead molecule (KdsA- lead1 docking complex) showed XP Gscore of ?10.26?kcal/mol and the binding interactions (Figure 2) were correlated favorably with PEP–KdsA interactions (Figure 1). Molecular dynamics simulations of KdsA– lead1 docking complex for 10?ns had revealed that the complex (Figure 3) remained stable in closer to physiological environmental condition. The predicted pharmacological properties of lead1 were well within the range of a drug molecule with good ADME profile, hence, would be intriguing towards development of potent inhibitor molecule against KdsA of Leptospira.     

3 Origins and evolution of the translation machinery

The protein synthesis machinery largely evolved prior to the last common ancestor and hence its study can provide insight to early events in the origin of life, including the transition from the hypothetical RNA world to living systems as we know them. By utilizing information from primary sequences, atomic resolution structures, and functional properties of the various components, it is possible to identify timing relationships (Hsiao et al., 2009; Fox, 2010). Taken together, these timing events are used to develop a preliminary time line for major evolutionary events leading to the modern protein synthesis machinery. It has been argued that a key initial event was the hybridization of two or more RNAs that created the peptidyl transferase center, (PTC), of the ribosome (Agmon et al. 2005). The PTC, left side of figure, contains a characteristic cavity/pore that serves as the entrance to the exit tunnel and is thought to be essential to the catalysis (Fox et al., 2012). This cavity is distinct from typical RNA pores (right side of figure) in that the nitrogenous bases face towards the lumen of the pore and thus are available for hydrogen bonding interactions. In typical RNA pores, the bases carefully avoid the lumen region. In support of Agmon et al. 2005), it is argued that this key difference reflects the fact the pore was created by an early hybridization event rather than normal RNA folding.     

136 Dendritic Alkyl Chains on DNA Cages: A Geometry-Dependent Inter- or Intramolecular “Handshake”

The selective association of hydrophobic sidechains is a strong determinant of protein organization. We have observed a parallel mode of assembly in DNA nanotechnology. Firstly, dendritic DNA amphiphiles (D-DNA) were synthesized (Carneiro, Aldaye, & Sleiman, 2009) comprising an addressable oligonucleotide portion and a hydrophobic alkyl dendron at the 5’ terminus. DNA amphiphiles have gathered interest recently as they can self-assemble in aqueous media to form well defined micelles while also retaining the ability to hybridize to their complement (Kwak & Herrmann, 2011; Patwa, et al. 2011) Two variations of alkyl D-DNA were hybridized to the single-stranded edges of a DNA cube (McLaughlin, et al., 2012). It was found that anisotropic organization of these hydrophobic domains on the 3D scaffold results in a new set of assembly rules, dependent on spatial orientation, number, and chemical identity of the D-DNA on the cubic structure (Edwardson. et al. 2012). When four amphiphiles are organized on one cube face, the hydrophobic residues engage in an intermolecular “handshake” between two cubes, resulting in a dimer. When eight amphiphiles are organized on the top and bottom faces of the cube, they engage in a “handshake” inside the cube. Combining the highly specific recognition of the oligonucleotide sequence with the orthogonal association of hydrophobic moieties can lead to a variety of structures with such diverse applications as membrane anchoring, cell uptake, directed hydrophobic assembly, and encapsulation and release of small molecules.     

160 A dynamic model for the linker histone

Linker histones play an important role in the packing of chromatin. This family of proteins generally consists of a short, unstructured N-terminal domain, a central globular domain, and a C-terminal domain (CTD). The CTD, which makes up roughly half of the protein, is intrinsically disordered in solution but adopts a specific fold upon interaction with DNA (Fang et al., 2012). While the globular domain structure is well characterized, the structure of the CTD remains unknown. Sequence alignment alone does not reveal any significant homologs for this region of the protein. Construction of a model thus requires additional information. For example, the atomic model for the rat histone H1d CTD, proposed over a decade ago, used novel bioinformatics tools and biochemical data (Bharath et al., 2002). New fluorescence resonance energy transfer (FRET) studies of the folding of the CTD in the presence of linear DNA, single nucleosomes, and oligonucleosomal arrays (Caterino et al., 2011; Fang et al., 2012) have stimulated our interest in constructing a dynamic model of the protein. We have obtained preliminary information about the structure and dynamics of the linker histone CTD through ab initio folding simulations using the Rosetta modeling package (Rohl et al., 2004). By analyzing a large number of conformations sampled through a Monte Carlo procedure, we get a clearer picture of the preferred states of the protein and its dynamics. Our results show that the CTD may frequently adopt a structure with 3–5 helices and helix-turn-helix motifs in specific regions. Some of the best scoring structures show high similarity with the HMG-box-containing proteins previously used as templates by Bharath et al. Further clustering analysis of our results hints of a preferred set of conformations for the CTD of the linker histone. Comparison of these models with distances measured by FRET may help account for the distinct structures of the CTD observed upon binding to different macromolecular partners.     

20 Supramolecular polymerization of nucleobase-like monomers in water

Elucidating the physiochemical principles that govern molecular self-assembly is of great importance for understanding biological systems and may provide insight into the emergence of the earliest macromolecules of life, an important challenge facing the RNA World hypothesis. Self-assembly results from a delicate balance between multiple noncovalent interactions and solvent effects, but achieving efficient self-assembly in aqueous solution with synthetic molecules has proven particularly challenging. Here, we demonstrate how two physical properties – monomer solubility and large hydrophobic surfaces of intermediate structures – are key elements to achieving supramolecular polymers in aqueous solution (Cafferty et al., 2013). Applying these two principles, we report the highly cooperative self-assembly of two weakly interacting, low molecular weight monomers [cyanuric acid and a modified triaminopyrimidine] into a water-soluble supramolecular assembly (see scheme below). The observed equilibrium between only two appreciably populated states – free monomers and supramolecular assemblies – is in excellent agreement with the values previously determined for the free energy of hydrogen bonding (Klostermeier & Millar, 2002), π???π stacking (Frier et al., 1985), and the calculated free energy penalty for the solvation of hydrophobic structures in water (Chandler, 2005). The similarity of the molecules used in this study for the nucleobases found in contemporary nucleic acids and the demonstration that these monomers assemble while the natural nucleobases do not, suggests that the first informational polymers may have emerged from a similar self-assembly process, if the nucleobases were different then they are today (Hud et al., 2013).     

69 DNA-binding studies of large antiviral polyamides

Polyamides are minor groove DNA-binding agents derived from the natural product distamycin A. PA1 is a large 12 ring polyamide discovered by NanoVir LLC; it is bioactive against the HPV16 virus in cell and tissue culture (Edwards et al., 2011). To better understand the basis of this phenomenon, the interactions of PA1 with the regulatory sequence of the HPV16 genome (7662–122?bp) are being examined. Using affinity cleavage as detected by capillary electrophoresis, with PA1 attached to methyl propyl ethidium iron EDTA, 10 binding sites of PA1 were identified in this part of the HPV genome. Polyamide perfect binding sites were as predicted by recognition rules (Dervan & Edelson, 2003). Quantitative DNaseI footprinting indicates that both perfect and single mismatch sites are bound with K_ds in the low nm range. Interestingly, a wide range of K_ds are observed for double mismatch sites (1–60?nm) and are under examination. This work will permit us to build a map of PA binding to HPV sequences, thus informing mechanisms of in vivo behavior.     

144 Sculpting light with DNA origami

We used the DNA origami method (Rothemund, 2006) for the fabrication of self-assembled nanoscopic materials (Seeman, 2010). In DNA origami, a virus-based 8?kilobase-long DNA single-strand is folded into shape with the help of ～ 200 synthetic oligonucleotides. The resulting DNA nanostructures can be designed to adopt any three-dimensional shape and can be addressed through DNA hybridization or chemical modification with nanometer precision. We have realized that complex assemblies of nanoparticles, including magnetic, fluorescent, and plasmonic nanoparticles. Such nanoconstructs may exhibit striking optical properties such as strong optical activity in the visible range (Kuzyk et al., 2012). To this end, plasmonic particles were assembled in solution to form helices of controlled handedness. We achieved spatial control over particle placement better than 2?nm and attachment yields of 97% and above. As a collective optical response emerging from our dispersed nanostructures, we detected pronounced circular dichroism (CD) originating from the plasmon–plasmon interactions in the particle helices. In recent experiments, we were able to show that the optical response of chiral biomolecules can be transferred from the UV into the visible region in plasmonic hotspots. Thus, sensitive detection of chiral biomolecules may become feasible in the near future. We also found that the orientation of the helices in respect to the incoming light beam critically influences the resulting CD spectra. Our results can be explained with theoretical models based on plasmonic dipole interaction and demonstrate the potential of DNA origami for the assembly of metafluids with designed optical properties.     

121 Influence of changes in DNA conformation on thermodynamic contribution during interaction of human genomic DNA and its mutant with anticancer drugs

Isothermal calorimetry (ITC) is efficient in characterizing and recognizing both high affinity and low affinity intermolecular interactions quickly and accurately. Adriamycin (ADR) and daunomycin (DNM) are the two anticancer drugs whose activity is achieved mainly by intercalation with DNA. During chemotherapy, normal human genomic DNA and mutated DNA from K562 leukemic cells show different thermodynamic properties and binding affinities on interaction with ADR and DNM when followed by ITC. Normal DNA shows more than one step in kinetic analysis, which could be attributed to outside binding, intercalation and reshuffling as suggested by Chaires et al. (1985); whereas K562 DNA fits a different binding pattern with higher binding affinities (by one order or more) compared to normal DNA. Structural properties of the interaction were followed by laser Raman spectroscopy, where difference in structure was apparent from the shifts in marker B DNA Raman bands (Ling et al., 2005). A correlation of thermodynamic contribution and structural data reveals step wise changes in normal genomic DNA conformation on drug binding. The overall structural change is higher in normal DNA–DNM interaction suggesting a partial B to A transition on drug binding. Such large changes were not observed for K562 DNA–DNM interaction which showed B to A transition properties in native from itself corroborating with our earlier findings (Ghosh et al., 2012).     

141 DNA origami as a platform for assembly of nanophotonic elements

Achieving DNA-functionalized semiconductor quantum dots (QDs) that are robust enough to be compatible with the DNA nanotechnology that withstand precipitation at high temperature and ionic strength is a challenge. Here we report a method that facilitates the synthesis of stable core/shell (1–20 monolayers) QD-DNA conjugates in which the end part (5–10 nucleotides) of the phosphorothiolated oligonucleotides is embedded within the shell of the QD. These reliable QD-DNA conjugates exhibit excellent chemical, colloidal and photonic stability over a wide pH range (4–12) and at high salt concentrations (>100?mM Na⁺ or Mg²⁺), bright fluorescence emission with quantum yields of upto 70%, and broad spectral tunability with emission ranging from UV to NIR (360–800?nm). The assembly of these different QDs into DNA origami in a well-controlled pattern was demonstrated (Deng, Samanta, Nangreave, Yan, & Liu, 2012). We also used DNA origami as a platform to co-assemble a gold nanoparticle with 20?nm diameter (AuNP) and an organic fluorophore (TAMRA) and studied the distance dependent plasmonic interactions between the particle and the dye using steady state fluorescence and lifetime measurements. Greater fluorescence quenching was found at smaller inter-particle distances, which was accompanied by an enhancement of the decay rate. We further fabricated 20?nm and 30?nm AuNP homodimers with different inter-particle distances using DNA origami scaffolds and positioned a Cy3 fluorophore between the AuNPs in both the assemblies. Up to 50% enhancement of the Cy3 fluorescence quantum efficiency was observed for the dye between the 30?nm AuNPs. These results are in good agreement with the theoretical simulations (Pal et al., 2013).     

Some progress in the study of plant–soil interactions in China

Plants and soil are the base for sustainably surviving human beings on the globe as the role of materials, energy, resources and environment (Shao & Chu 2008; Shao et al. 2008, 2009, 2010, 2012a,b; Liu & Shao, 2010; Ruan et al. 2010; Xu et al. 2010, 2012; Shao 2012; Huang et al. 2013). This topic has been extensively investigated for 100 years with more achievements in many sectors and practical significance in conducting high-efficient agriculture and eco-environmental construction. The plant–soil interaction is the core issue of this topic, which has been given much attention for the past 30 years (Wu et al. 2007, 2010; Zhang et al. 2011, 2013; Xu et al. 2012, 2013).     

177 T-cell vaccine design for Streptococcus pneumoniae: an in silico approach

Streptococcus pneumoniae (pneumococcus) remains an important cause of meningitis, bacteremia, acute otitis media, community acquired pneumonia associated with significant morbidity, and mortality world wide. Conjugated polysaccharide, glycoconjugated, and capsular polysaccharide based vaccines were existent for pneumococcal disease but are still specific and restricted to serotypes of S. pneumoniae. Proteome of eight serotypes of S. pneumoniae was retrieved and identified in common proteins (Munikumar et al., 2012). 18 membrane proteins were distinguished from 1657 common proteins of eight serotypes of S. pneumoniae. Implementing comparative genomic approach and subtractive genomic approach, three membrane proteins were predicted as essential for bacterial survival and non-homologous to human (Munikumar et al., 2012; Umamaheswari et al., 2011). ProPred server was used to propose four promiscuous T-cell epitopes from three membrane proteins and validated through published positive control, SYFPEITHI and immune epitope database (Munikumar et al., in press). The four epitopes docked into peptide binding region of predominant HLA-DRB alleles with good binding affinity in Maestro v9.2. The T-cell epitope 89-VVYLLPILI-97 and HLA-DRB5^?0101 docking complex was with best XPG score (?13.143?kcal/mol). Further, the stability of the complex was checked through molecular dynamics simulations in Desmond v3.3. The simulation results had revealed that the complex was stable throughout 5000?ps (Munikumar et al., in press). Thus, the epitope would be the ideal candidate for T-cell driven subunit vaccine design against selected serotypes of S. pneumoniae.     

167 Network properties of decoy and CASP predicted models: a comparison with native protein structures

Principles that govern protein folding still remain elusive. Given the huge sequence space, it is reasonable to assume that sequences follow a particular pattern to attain one of the folds already defined in the relatively small structural space. In this study, we have used protein structure networks at different interaction strengths of non-covalent interactions (I_min) (Brinda & Vishveshwara, 2005; Kannan & Vishveshwara, 1999), to identify patterns that can distinguish a native protein from decoy/modelled structures. This is a rigorous extension of an earlier study performed at I_min???0% (Chatterjee, Bhattacharyya et al., 2012). Network properties such as the size of the largest cluster (SLClu), largest k-2 communities (ComSk2) and clustering coefficients (CCoe) are analysed for 5422 native structures and 29543 decoy/modelled structures. Steeper transition profile of the native structures as a function of I_min is consistently observed (see Figure) . The network properties generated at different I_min and main-chain hydrogen bonds (MHB) are integrated into support vector machine to build a classifier, giving an accuracy of 94.11%. The uniqueness of the protein structures through side-chain interactions are captured by the network parameters, while MHB represents the backbone packing. Quality predictions for the recently concluded CASP 10 predicted models are also performed using the model with the selected ones showing RMSD values?<?2.5?Å with respect to the native structures. Amongst the network properties, ComSk2 is maximally able to capture the transition properties of the structures. Importance of ComSk2 has earlier been implicated to capture the percolating behaviour of a protein structure (Deb & Vishveshwara, 2009). Overall, a robust classifier is obtained, and patterns specific to native structures have been analysed and discussed. The study highlights the importance of side-chain interactions at different I_mins, along with backbone level interactions.     

128 Some derivatives of 1,3-diazaadamantane strongly stimulate strand exchange reaction between short oligonucleotides

Earlier, we characterized the behavior of HG122 (compound III) in the DNA Strand Exchange Reaction (SER) and found that 1,3-diazaadamantane derivative facilitates SER in the system of short oligonucleotides (Gabrielian et al., 2011). In the present study, a series of new derivatives of 1,3-diazaadamantane have been synthesized with the purpose to discern how small variations in the compound structure can influence its activity in SER and try to get more effective substances for stimulation of SER. We revealed that most of the small variations in the structure significantly influenced the compounds’ efficacy in accelerating SER. For example, an increase in the compounds’ aliphatic chain lengths considerably enhanced its efficiency in SER stimulation and in the series of compounds presented in the Figure HG188 (compound IV) was eminently the most potent agent in the stimulation of SER. Small modifications in other parts of the 1,3-diazaadamantane molecule also influenced the SER stimulation and several derivatives more efficient in facilitating SER than HG122 were revealed. Some of the compounds exhibited virtually negligible capability to stimulate SER but, interestingly, out of 12 derivatives characterized, agents that retard spontaneous SER were not found. Earlier, the stimulation of the DNA strand exchange was documented for different ligands of the policationic nature such as Cationic Comb Copolymers (Kim et al., 2003), linker histones (Bocharova et al., 2012), cobalt hexamine (unpublished observation) etc. The present results provide us with a novel class of SER facilitating compounds – the cationic amphiphiles that can serve as interesting objects for further understanding of different aspects of DNA SER. Biomedical implications and prospects in biomedical applications of these and similar compounds’ activity in SER remain to be investigated.     

172 Role of conserved water molecular triad in the recognition of IMP,NAD+ with Asp 274, Asn 303, Arg 322, and Asp 364 in both the isoform of hIMPDH

Inosine monophosphate dehydrogenase (IMPDH) plays an important role in the Guanosine monophosphate (GMP) biosynthesis pathway. As hIMPDH-II is involved in CML-Cancer, it is thought to be an active target for leukemic drug design. The importance of conserved water molecules in the salt-bridge-mediated interdomain recognition and loop-flap recognition of hIMPDH has already been indicated in some simulation studies (Bairagya et al., 2009, 2011a, 2011b, 2012; Mishra et al., 2012). In this work, the role of conserved water molecules in the recognition of Inosine monophosphate (IMP) and NAD⁺ (co-factor) to active site residues of both the isoforms has been investigated by all atoms MD-Simulation studies. During 25-ns dynamics of the solvated hIMPDH-II and I (1B3O and 1JCN PDB structures), the involvement of conserved water molecular triad (W _M, W _L and W _C) in the recognition of active site residues (Asp 274, Asn 303, Arg 322, and Asp 364), IMP and NAD⁺ has been observed (Figure 1). The H-bonding co-ordination of all three conserved water molecular centers is within 4–7 and their occupation frequency is 1.0. The H-bonding geometry and the electronic consequences of the water molecular interaction at the different residues (and also IMP and NAD⁺) may put forward some rational clues on antileukemic agent design.