Solid state stacked trimers of 1,10-phenanthroline in a supramolecular tubular motif of C-methyl calix[4]resorcinarene

The self-assembly of bidentate ligand, 1,10-phenanthroline with C-methyl calix[4]resorcinarene (CMCR) in presence of coumarin results in a unique trimer stacking arrangement of phenanthroline molecules in a nanotubular motif generated by the supramolecular assembly of the host.

The poly dA helix: a new structural motif for high performance DNA-based molecular switches

We report a pH-dependent conformational transition in short, defined homopolymeric deoxyadenosines (dA(15)) from a single helical structure with stacked nucleobases at neutral pH to a double-helical, parallel-stranded duplex held together by AH-HA base pairs at acidic pH. Using native PAGE, 2D NMR, circular dichroism (CD) and fluorescence spectroscopy, we have characterized the two different pH dependent forms of dA(15). The pH-triggered transition between the two defined helical forms of dA(15) is characterized by CD and fluorescence. The kinetics of this conformational switch is found to occur on a millisecond time scale. This robust, highly reversible, pH-induced transition between the two well-defined structured states of dA(15)represents a new molecular building block for the construction of quick-response, pH-switchable architectures in structural DNA nanotechnology.

Observation of a mixed antiparallel and parallel beta-sheet motif in the crystal structure of Boc-Ala-Ile-Aib-OMe

A diastereomeric mixture of the tripeptide Boc-Ala-Ile-Aib-OMe crystallized in the space group P1 from CH3OH/H2O. The unit cell parameters are a = 10.593(2) A, b = 14.377(3) A, c = 17.872(4) A, alpha = 104.41(2) degrees, beta = 90.55(2) degrees, gamma = 106.91(2) degrees, V = 2512.4 A3, Z = 4. X-Ray crystallographic studies show the presence of four molecules in the asymmetric unit consisting of two pairs of diastereomeric peptides, Boc-L-Ala-L-Ile-Aib-OMe and Boc-L-Ala-D-Ile-Aib-OMe. The four molecules in the asymmetric unit form a rarely found mixed antiparallel and parallel beta-sheet hydrogen bond motif. The Ala and (L,D)-Ile residues in all the four molecules adopt the extended conformations, while the phi, psi values of the Aib residues are in the right-handed helical region. In one of the molecules the Ile sidechain adopts the unusual gauche conformation about the C beta-C gamma bond.

Deciphering the Distinct Role for the Metal Coordination Motif in the Catalytic Activity of Mycobacterium smegmatis Topoisomerase I

Mycobacterium smegmatis topoisomerase I (Mstopol) is distinct from typical type IA topoisomerases. The enzyme binds to both single- and double-stranded DNA with high affinity, making specific contacts. The enzyme comprises conserved regions similar to type IA topoisomerases from Escherichia coli and other eubacteria but lacks the typically found zinc fingers in the carboxy-terminal domain. The enzyme can perform DNA cleavage m the absence of Mg2+ but religation needs exogenously added Mg2+. One molecule of Mg2+ tightly bound to the enzyme has no role in DNA cleavage but is needed only for the religation reaction. The toprim. (topoisomerase-primase) domain in MstopoI comprising the Mg2+ binding pocket, conserved in both type IA and type II topoisomerases, was subjected to mutagenesis to understand the role of Mg2+, in different steps of the reaction. The residues D108, D110, and E112 of the enzyme, which form the acidic triad in the DXDXE motif, were changed to alanines. D108A mutation resulted in an enzyme that is Mg2+ dependent for DNA cleavage unlike Mstopol and exhibited enhanced DNA cleavage property and reduced religation activity. The mutant was toxic for cell growth, most likely due to the imbalance in cleavage-religation equilibrium. In contrast, the E112A mutant behaved like wild-type enzyme, cleaving DNA in a Mg2+-independent fashion, albeit to a reduced extent. Intra- and intermolecular religation assays indicated specific roles for D108 and E112 residues during the reaction. Together, these results indicate that the D108 residue has a major role during cleavage and religation, while E112 is important for enhancing the efficiency of cleavage. Thus, although architecturally and mechanistically similar to topoisomerase I from E. coli, the metal coordination pattern of the mycobacterial enzyme is distinct, opening up avenues to exploit the enzyme to develop inhibitors.

Modulating the preferred O-H center dot center dot center dot O hydrogen-bonding motif in a conformationally constrained environment through hydroxy-group derivatization

The crystal structures of three conformationally locked esters, namely the centrosymmetric tetrabenzoate of all-axial per-hydronaphthalene- 2,3,4a, 6,7,8a-hexaol, viz. trans-4a, 8a-dihydroxyperhydronaphthalene-2,3,6,7-tetrayl tetrabenzoate, C38H34O10, and the diacetate and dibenzoate of all-axial perhydronaphthalene-2,3,4a, 8a-tetraol, viz. (2R*,3R*,4aS*,8aS*)-4a, 8a-dihydroxyperhydronaphthalene-2,3-diyl diacetate, C-14-H22O6, and (2R*, 3R*, 4aS*, 8aS*)-4a, 8a-dihydroxyperhydronaphthalene- 2,3-diyl dibenzoate, C24H26O6, have been analyzed in order to examine the preference of their supramolecular assemblies towards competing inter-and intramolecular O-H center dot center dot center dot O hydrogen bonds. It was anticipated that the supramolecular assembly of the esters under study would adopt two principal hydrogen-bonding modes, namely one that employs intermolecular O-H center dot center dot center dot O hydrogen bonds (mode 1) and another that sacrifices those for intramolecular O-H center dot center dot center dot O hydrogen bonds and settles for a crystal packing dictated by weak intermolecular interactions alone (mode 2). Thus, while the molecular assembly of the two crystalline diacyl derivatives conformed to a combination of hydrogen-bonding modes 1 and 2, the crystal packing in the tetrabenzoate preferred to follow mode 2 exclusively.

Probing Fluorine Interactions in a Polyhydroxylated Environment: Conservation of a C-F center dot center dot center dot H-C Recognition Motif in Presence of O-H center dot center dot center dot O Hydrogen Bonds

Three conformationally locked fluorinated polycyclitols have been specially crafted on a rigid trans-decalin backbone, employing a surprisingly facile pyridine-poly(hydrogen fluoride)-mediated stereospecific epoxide ring opening as the key reaction. Molecula design of the three fluorinated probes under study focused on providing an efficient platform for (a) evaluating the ability of covalently bonded fluorine, vis-a-vis the isosteric hydroxy group, to act as a H-bond acceptor and (b) examining the possibility for an organic fluorine moiety, placed suitably in a spatially invariant position, to engage an 1,3-diaxial OH functionality in a purported intramolecular O-H center dot center dot center dot F hydrogen bond. The present endeavour reveals that C(sp(3))-F center dot center dot center dot H-C(sp(3)) hydrogen bonds, though weak and lesser investigated, can indeed be observed and supramolecular recognition motifs, involving such interactions, can be conserved even in crystal structures laden with stronger O-H center dot center dot center dot O hydrogen bonds.

A C-H…O hydrogen bond stabilized polypeptide chain reversal motif at the C-terminus helices in proteins.

The serendipitous observation of a C–Hcdots, three dots, centeredO hydrogen bond mediated polypeptide chain reversal in synthetic peptide helices has led to a search for the occurrence of a similar motif in protein structures. From a dataset of 634 proteins, 1304 helices terminating in a Schellman motif have been examined. The C–Hcdots, three dots, centeredO interaction between the T−4 CαH and T+1 C=O group (Ccdots, three dots, centeredO≤3.5 Å) becomes possible only when the T+1 residue adopts an extended β conformation (T is defined as the helix terminating residue adopting an αL conformation). In all, 111 examples of this chain reversal motif have been identified and the compositional and conformational preferences at positions T−4, T, and T+1 determined. A marked preference for residues like Ser, Glu and Gln is observed at T−4 position with the motif being further stabilized by the formation of a side-chain–backbone Ocdots, three dots, centeredH–N hydrogen bond involving the side-chain of residue T−4 and the N–H group of residue T+3. In as many as 57 examples, the segment following the helix was extended with three to four successive residues in β conformation. In a majority of these cases, the succeeding β strand lies approximately antiparallel with the helix, suggesting that the backbone C–Hcdots, three dots, centeredO interactions may provide a means of registering helices and strands in an antiparallel orientation. Two examples were identified in which extended registry was detected with two sets of C–Hcdots, three dots, centeredO hydrogen bonds between (T−4) CαHcdots, three dots, centeredC=O (T+1) and (T−8) CαHcdots, three dots, centeredC=O (T+3).

Tribridged diruthenium complexes: A new structural motif

Dinuclear complexes containing a (mu-oxo)bis(mu-carboxylato) diruthenium (III) core have been prepared by a novel synthetic route using metal-metal bonded diruthenium(II,III) tetracarboxylates as precursors. The complexes have been structurally characterized and they are redox active. The terminal ligands play an important role in tuning the electronic structure of the core. The stability of the core is found to be dependent on the size and pi-acidic nature of the terminal ligand cis- to the mu-oxo ligand. The chemistry of such tribridged complexes is relatively new. The rapid growth of this chemistry is based on the discovery of similar core structures in several non-heme iron- and manganese-containing metalloproteins. The tribridged core presents a new structural motif in coordination chemistry. The chemistry of diruthenium complexes with a [Ru-2(mu-O) (mu-O(2)CR)(2)(2+)] core has been reviewed.

Preferential condensation of SAR-DNA by histone H1 and its SPKK containing octapeptide repeat motif

Linker histone H1 binds preferentially the scaffold associated region (SAR) DNA elements that contain characteristic oligo dA . dT tracts. In the present study, we have compared the condensation brought about by histone H1 of a SAR DNA fragment in the histone spacer region of Drosophila melanogaster with that of a random DNA (pBR322 EcoRI-SalI) fragment by circular dichroism spectroscopy. The condensation of the SAR DNA fragment by histone H1 is 3-4-fold higher than that of the random DNA fragment. A 16-mer peptide, ATPKKSTKKTPKKAKK, the sequence that is present in the C-terminus of histone H1d, which has recently been shown to possess DIVA and chromatin condensing properties, also condenses the SAR DNA fragment preferentially in a highly cooperative manner. We have proposed a model for the dynamics of chromatin structure involving histone H1-SAR DNA interaction through SPKK containing peptide motifs and its competition by AT-hook peptides present in the nonhistone chromosomal proteins like HMG-I and HMG-Y.

A C-H center dot center dot center dot O hydrogen bond stabilized polypeptide chain reversal motif at the C terminus of helices in proteins

The serendipitous observation of a C-H...O hydrogen bond mediated polypeptide chain reversal in synthetic peptide helices has led to a search for the occurrence of a similar motif in protein structures. From a dataset of 634 proteins, 1304 helices terminating in a Schellman motif have been examined. The C-H...O interaction between the T - 4 (CH)-H-alpha and T + 1 C=O group (C...O 3.5 Angstrom) becomes possible only when the T + 1 residue adopts an extended beta conformation (T is defined as the helix terminating residue adopting an alpha(L) conformation). In all, 111 examples of this chain reversal motif have been identified and the compositional and conformational. preferences at positions T - 4, T, and T + 1 determined. A marked preference for residues like Set, Glu and Gln is observed at T - 4 position with the motif being further stabilized by the formation of a side-chain-backbone O...H-N hydrogen bond involving the side-chain of residue T - 4 and the N-H group of residue T + 3. In as many as 57 examples, the segment following the helix was extended with three to four successive residues in beta conformation. In a majority of these cases, the succeeding beta strand lies approximately antiparallel with the helix, suggesting that the backbone C-H...O interactions may provide a means of registering helices and strands in an antiparallel orientation. Two examples were identified in which extended registry was detected with two sets of C-H...O hydrogen bonds between (T - 4) (CH)-H-alpha...C=O (T + 1) and (T - 8) (CH)-H-alpha...C=O (T + 3). 0 2002 Published by Elsevier Science Ltd.

Role of Specific Cations and Water Entropy on the Stability of Branched DNA Motif Structures

DNA three-way junctions (TWJs) are important intermediates in various cellular processes and are the simplest of a family of branched nucleic acids being considered as scaffolds for biomolecular nanotechnology. Branched nucleic acids are stabilized by divalent cations such as Mg2+, presumably due to condensation and neutralization of the negatively charged DNA backbone. However, electrostatic screening effects point to more complex solvation dynamics and a large role of interfacial waters in thermodynamic stability. Here, we report extensive computer simulations in explicit water and salt on a model TWJ and use free energy calculations to quantify the role of ionic character and strength on stability. We find that enthalpic stabilization of the first and second hydration shells by Mg2+ accounts for 1/3 and all of the free energy gain in 50% and pure MgCl2 solutions, respectively. The more distorted DNA molecule is actually destabilized in pure MgCl2 compared to pure NaCl. Notably, the first shell, interfacial waters have very low translational and rotational entropy (i.e., mobility) compared to the bulk, an entropic loss that is overcompensated by increased enthalpy from additional electrostatic interactions with Mg2+. In contrast, the second hydration shell has anomalously high entropy as it is trapped between an immobile and bulklike layer. The nonmonotonic entropic signature and long-range perturbations of the hydration shells to Mg2+ may have implications in the molecular recognition of these motifs. For example, we find that low salt stabilizes the parallel configuration of the three-way junction, whereas at normal salt we find antiparallel configurations deduced from the NMR. We use the 2PT analysis to follow the thermodynamics of this transition and find that the free energy barrier is dominated by entropic effects that result from the decreased surface area of the antiparallel form which has a smaller number of low entropy waters in the first monolayer.

Ca2+ Binding to the ExDxD Motif Regulates the DNA Cleavage Specificity of a Promiscuous Endonuclease

Most of the restriction endonucleases (REases) are dependent on Mg2+ for DNA cleavage, and in general, Ca2+ inhibits their activity. RKpnI, an HNH active site containing beta beta alpha-Me finger nuclease, is an exception. In presence of Ca2+, the enzyme exhibits high-fidelity DNA cleavage and complete suppression of Mg2+-induced promiscuous activity. To elucidate the mechanism of unusual Ca2+-mediated activity, we generated alanine variants in the putative Ca-2+ binding motif, E(132)xD(134)xD(136), of the enzyme. Mutants showed decreased levels of DNA cleavage in the presence of Ca2+. We demonstrate that ExDxD residues are involved in Ca2+ coordination; however, the invariant His of the catalytic HNH motif acts as a general base for nucleophile activation, and the other two active site residues, D148 and Q175, also participate in Ca2+-mediated cleavage. Insertion of a 10-amino acid linker to disrupt the spatial organization of the ExDxD and HNH motifs impairs Ca2+ binding and affects DNA cleavage by the enzyme. Although ExDxD mutant enzymes retained efficient cleavage at the canonical sites in the presence of Mg2+, the promiscuous activity was greatly reduced, indicating that the carboxyl residues of the acidic triad play an important role in sequence recognition by the enzyme. Thus, the distinct Ca2+ binding motif that confers site specific cleavage upon Ca2+ binding is also critical for the promiscuous activity of the Mg2+-bound enzyme, revealing its role in metal ion-mediated modulation of DNA cleavage.

Cloning, expression, purification, and biochemical characterisation of the FIC motif containing protein of Mycobacterium tuberculosis

The role of FIC (Filamentation induced by cAMP)(2) domain containing proteins in the regulation of many vital pathways, mostly through the transfer of NMPs from NTPs to specific target proteins (NMPylation), in microorganisms, higher eukaryotes, and plants is emerging. The identity and function of FIC domain containing protein of the human pathogen, Mycobacterium tuberculosis, remains unknown. In this regard, M. tuberculosis fic gene (Mtfic) was cloned, overexpressed, and purified to homogeneity for its biochemical characterisation. It has the characteristic FIC motif, HPFREGNGRSTR (HPFxxGNGRxxR), spanning 144th to 155th residue. Neither the His-tagged nor the GST-tagged MtFic protein, overexpressed in Escherichia coil, nor expression of Mtfic in Mycobacterium smegmatis, yielded the protein in the soluble fraction. However, the maltose binding protein (MBP) tagged MtFic (MBP-MtFic) could be obtained partly in the soluble fraction. The cloned, overexpressed, and purified recombinant MBP-MtFic showed conversion of ATP, GTP, CTP, and UTP into AMP. GMP, CMP, and UMP, respectively. Sequence alignment with several FIC motif containing proteins, complemented with homology modeling on the FIC motif containing protein, VbhT of Bartonella schoenbuchensis as the template, showed conservation and interaction of residues constituting the FIC domain. Site-specific mutagenesis of the His144, or Glu148, or Asn150 of the FIC motif, or of Arg87 residue that constitutes the FIC domain, or complete deletion of the FIC motif, abolished the NTP to NMP conversion activity. The design of NMP formation assay using the recombinant, soluble MtFic would enable identification of its target substrate for NMPylation. (C) 2012 Elsevier Inc. All rights reserved.