Interaction of palladium (II) with DNA

The nature of interaction of palladium(II) with calf thymus DNA was studied using viscometry, ultraviolet, visible and infrared spectrophotometry and optical rotatory disperison and circular dichroism measurements. The results indicate that Pd(II) interacts with both the phosphate and bases of DNA. The ORD/CD data indicate that the binding of Pd(II) to DNA brings about considerable conformational changes in DNA.

Interaction of DNA with anti-cancer drugs: copper-thiosemicarbazide system

The interaction of copper-thiosemicarbazide complexes with DNA was investigated using ultraviolet and infrared spectroscopy. Evidence for the interaction of the complexes with nucleic acid bases and with the phosphate group is presented.

A novel intermediate in the interaction of thiosemicarbazide with sheep liver serine hydroxymethyltransferase

An unusual intermediate bound to the enzyme was detected in the interaction of thiosemicarbazide with sheep liver serine hydroxymethyltransferase. This intermediate had absorbance maxima at 464 and 440 nm. Such spectra are characteristic of resonance stabilized intermediates detected in the interaction of substrates and quasi-substrates with pyridoxal phosphate enzymes. An intermediate of this kind has not been detected in the interaction of thiosemicarbazide with other pyridoxal phosphate enzymes. This intermediate was generated slowly (t 1/2 = 4 min) following the addition of thiosemicarbazide (200 microM) to sheep liver serine hydroxymethyltransferase (5 microM). It was bound to the enzyme as evidenced by circular dichroic bands at 464 and 440 nm and the inability to be removed upon Centricon filtration. The kinetics of interaction revealed that thiosemicarbazide was a slow binding reversible inhibitor in this phase with a k(on) of 11 M-1 s-1 and a k(off) of 5 x 10(-4) s-1. The intermediate was converted very slowly (k = 4 x 10(-5) s-1) to the final products, namely the apoenzyme and the thiosemicarbazone of pyridoxal phosphate. A minimal kinetic mechanism involving the initial conversion to the intermediate absorbing at longer wavelengths and the conversion of this intermediate to the final product, as well as, the formation of pyridoxal phosphate-thiosemicarbazone directly by an alternate pathway is proposed.

Interaction of surfactants with DNA. Role of hydrophobicity and surface charge on intercalation and DNA melting

A probe, 9-(anthrylmethyl)trimethylammonium chloride, 1, was prepared. 1 binds to calf-thymus DNA or Escherichia coli genomic DNA with high affinity, as evidenced from the absorption titration. Strong hypochromism, spectral broadening and red-shifts in the absorption spectra were observed. Half-reciprocal plot constructed from this experiment gave binding constant of 5±0.5×104 M−1 in base molarity. We employed this anthryl probe-DNA complex for studying the effects of addition of various surfactant to DNA. Surfactants of different charge types and chain lengths were used in this study and the effects of surfactant addition to such probe-DNA complex were compared with that of small organic cations or salts. Addition of either salts or cationic surfactants led to structural changes in DNA and under these conditions, the probe from the DNA-bound complex appeared to get released. However, the cationic surfactants could induce such release of the probe from the probe-DNA complex at a much lower concentration than that of the small organic cations or salts. In contrast the anionic surfactants failed to promote any destabilization of such probe-DNA complexes. The effects of additives on the probe-DNA complexes were also examined by using a different technique (fluorescence spectroscopy) using a different probe ethidium bromide. The association complexes formed between the cationic surfactants and the plasmid DNA pTZ19R, were further examined under agarose gel electrophoresis and could not be visualized by ethidium bromide staining presumably due to cationic surfactant-induced condensation of DNA. Most of the DNA from such association complexes can be recovered by extraction of surfactants with phenol-chloroform. Inclusion of surfactants and other additives into the DNA generally enhanced the DNA melting temperatures by a few °C and at high [surfactant], the corresponding melting profiles got broadened.

Molecular modeling of Bt Cry1Ac (DI–DII)–ASAL (Allium sativum lectin)–fusion protein and its interaction with aminopeptidase N (APN) receptor of Manduca sexta

Genetic engineering of Bacillus thuringiensis (Bt) Cry proteins has resulted in the synthesis of various novel toxin proteins with enhanced insecticidal activity and specificity towards different insect pests. In this study, a fusion protein consisting of the DI–DII domains of Cry1Ac and garlic lectin (ASAL) has been designed in silico by replacing the DIII domain of Cry1Ac with ASAL. The binding interface between the DI–DII domains of Cry1Ac and lectin has been identified using protein–protein docking studies. Free energy of binding calculations and interaction profiles between the Cry1Ac and lectin domains confirmed the stability of fusion protein. A total of 18 hydrogen bonds was observed in the DI–DII–lectin fusion protein compared to 11 hydrogen bonds in the Cry1Ac (DI–DII–DIII) protein. Molecular mechanics/Poisson–Boltzmann (generalized-Born) surface area [MM/PB (GB) SA] methods were used for predicting free energy of interactions of the fusion proteins. Protein–protein docking studies based on the number of hydrogen bonds, hydrophobic interactions, aromatic–aromatic, aromatic–sulphur, cation–pi interactions and binding energy of Cry1Ac/fusion proteins with the aminopeptidase N (APN) of Manduca sexta rationalised the higher binding affinity of the fusion protein with the APN receptor compared to that of the Cry1Ac–APN complex, as predicted by ZDOCK, Rosetta and ClusPro analysis. The molecular binding interface between the fusion protein and the APN receptor is well packed, analogously to that of the Cry1Ac–APN complex. These findings offer scope for the design and development of customized fusion molecules for improved pest management in crop plants.

Calculations of the free energy of interaction of the c-Fos−c-Jun coiled coil: Effects of the solvation model and the inclusion of polarization effects

The leucine zipper region of activator protein-1 (AP-1) comprises the c-Jun and c-Fos proteins and constitutes a well-known coiled coil protein−protein interaction motif. We have used molecular dynamics (MD) simulations in conjunction with the molecular mechanics/Poisson−Boltzmann generalized-Born surface area [MM/PB(GB)SA] methods to predict the free energy of interaction of these proteins. In particular, the influence of the choice of solvation model, protein force field, and water potential on the stability and dynamic properties of the c-Fos−c-Jun complex were investigated. Use of the AMBER polarizable force field ff02 in combination with the polarizable POL3 water potential was found to result in increased stability of the c-Fos−c-Jun complex. MM/PB(GB)SA calculations revealed that MD simulations using the POL3 water potential give the lowest predicted free energies of interaction compared to other nonpolarizable water potentials. In addition, the calculated absolute free energy of binding was predicted to be closest to the experimental value using the MM/GBSA method with independent MD simulation trajectories using the POL3 water potential and the polarizable ff02 force field, while all other binding affinities were overestimated.

Role of Arg-401 of cytosolic serine hydroxymethyltransferase in subunit assembly and interaction with the substrate carboxy group.

In an attempt to identify the arginine residue involved in binding of the carboxylate group of serine to mammalian serine hydroxymethyltransferase, a highly conserved Arg-401 was mutated to Ala by site-directed mutagenesis. The mutant enzyme had a characteristic visible absorbance at 425 nm indicative of the presence of bound pyridoxal 5'-phosphate as an internal aldimine with a lysine residue. However, it had only 0.003% of the catalytic activity of the wild-type enzyme. It was also unable to perform reactions with glycine, beta-phenylserine or d-alanine, suggesting that the binding of these substrates to the mutant enzyme was affected. This was also evident from the interaction of amino-oxyacetic acid, which was very slow (8.4x10(-4) s-1 at 50 microM) for the R401A mutant enzyme compared with the wild-type enzyme (44.6 s-1 at 50 microM). In contrast, methoxyamine (which lacks the carboxy group) reacted with the mutant enzyme (1.72 s-1 at 250 microM) more rapidly than the wild-type enzyme (0.2 s-1 at 250 microM). Further, both wild-type and the mutant enzymes were capable of forming unique quinonoid intermediates absorbing at 440 and 464 nm on interaction with thiosemicarbazide, which also does not have a carboxy group. These results implicate Arg-401 in the binding of the substrate carboxy group. In addition, gel-filtration profiles of the apoenzyme and the reconstituted holoenzyme of R401A and the wild-type enzyme showed that the mutant enzyme remained in a tetrameric form even when the cofactor had been removed. However, the wild-type enzyme underwent partial dissociation to a dimer, suggesting that the oligomeric structure was rendered more stable by the mutation of Arg-401. The increased stability of the mutant enzyme was also reflected in the higher apparent melting temperature (Tm) (61 degrees C) than that of the wild-type enzyme (56 degrees C). The addition of serine or serinamide did not change the apparent Tm of R401A mutant enzyme. These results suggest that the mutant enzyme might be in a permanently 'open' form and the increased apparent Tm could be due to enhanced subunit interactions.

Analysis of human chorionic gonadotropin-monoclonal antibody interaction in BIAcore

Kinetic studies of macromolecular ligand-ligate interaction have generated ample interest since the advent of plasmon resonance based instruments like BIAcore. Most of the studies reported in literature assume a simple 1 : 1 Langmuir binding and complete reversibility of the system. However we observed that in a high affinity antigen-antibody system [human chorionic gonadotropin-monoclonal antibody (hCG-mAb)] dissociation is insignificant and the sensogram data cannot be used to measure the equilibrium and kinetic parameters. At low concentrations of mAb the complete sensogram could be fitted to a single exponential. Interestingly we found that at higher mAb concentrations, the binding data did not conform to a simple bimolecular model. Instead, the data fitted a two-step model, which may be because of surface heterogeneity of affinity sites. In this paper, we report on the global fit of the sensograms. We have developed a method by which a single two-minute sensogram can be used in high affinity systems to measure the association rate constant of the reaction and the functional capacity of the ligand (hCG) immobilized on the chip. We provide a rational explanation for the discrepancies generally observed in most of the BIAcore sensograms

A study of interaction in the lowest singlet and triplet states of H2

The potential energy curves of the ground state and the first excited state of H2 are examined in terms of the electronic force acting on each nucleus. The results reveal the detailed course of events that occur when two hydrogen atoms with parallel and antiparallel electron spins approach one another from a large internuclear separation.

Acetylation of Transition Protein 2 (TP2) by KAT3B (p300) Alters Its DNA Condensation Property and Interaction with Putative Histone Chaperone NPM3

The hallmark of mammalian spermiogenesis is the dramatic chromatin remodeling process wherein the nucleosomal histones are replaced by the transition proteins TP1, TP2, and TP4. Subsequently these transition proteins are replaced by the protamines P1 and P2. Hyperacetylation of histone H4 is linked to their replacement by transition proteins. Here we report that TP2 is acetylated in vivo as detected by anti-acetylated lysine antibody and mass spectrometric analysis. Further, recombinant TP2 is acetylated in vitro by acetyltransferase KAT3B (p300) more efficiently than by KAT2B (PCAF). In vivo p300 was demonstrated to acetylate TP2. p300 acetylates TP2 in its C-terminal domain, which is highly basic in nature and possesses chromatin-condensing properties. Mass spectrometric analysis showed that p300 acetylates four lysine residues in the C-terminal domain of TP2. Acetylation of TP2 by p300 leads to significant reduction in its DNA condensation property as studied by circular dichroism and atomic force microscopy analysis. TP2 also interacts with a putative histone chaperone, NPM3, wherein expression is elevated in haploid spermatids.Interestingly, acetylation of TP2 impedes its interaction with NPM3. Thus, acetylation of TP2 adds a new dimension to its role in the dynamic reorganization of chromatin during mammalian spermiogenesis.

Outer Membrane Protease/Adhesin PgtE of Salmonella enterica: Role in Salmonella-Host Interaction

Salmonella enterica serovar Typhimurium is a common cause of gastroenteritis in humans and, occasionally, also causes systemic infection. During systemic infection an important characteristic of Salmonella is its ability to survive and replicate within macrophages. The outer membrane protease PgtE of S. enterica is a member of the omptin family of outer membrane aspartate proteases, which are beta-barrel proteins with five surface-exposed loops. The main goals of this study were to characterize biological substrates and pathogenesis-associated functions of PgtE and to determine the conditions where PgtE is fully active. In this study we found that PgtE requires rough lipopolysaccharide (LPS) to be functional but is sterically inhibited by the long O-antigen side chain in smooth LPS. Salmonella isolates normally are smooth with a long oligosaccharide O-antigen, and PgtE remains functionally cryptic in wild-type Salmonella cultivated in vitro. Interestingly, our results showed that due to increased expression of PgtE and to reduced length of the LPS O-antigen chains, the wild-type Salmonella expresses highly functional PgtE when isolated from mouse macrophage-like J774A.1 cells. Salmonella is thought to be continuously released from macrophages to infect new ones, and our results suggest that PgtE is functional during these transient extracellular growth phases. Six novel host protein substrates were identified for PgtE in this work. PgtE was previously known to activate human plasminogen (Plg) to plasmin, a broad-spectrum serine protease, and in this study PgtE was shown to interfere with the Plg system by inactivating the main inhibitor of plasmin, alpha2-antiplasmin. PgtE also interferes with another important proteolytic system of mammals by activating pro-matrix metalloproteinase-9 to an active gelatinase. PgtE also directly degrades gelatin, a component of extracellular matrices. PgtE also increases bacterial resistance against complement-mediated killing in human serum and enhances survival of Salmonella within murine macrophages as well as in the liver and spleen of intraperitoneally infected mice. Taken together, the results in this study suggest that PgtE is a virulence factor of Salmonella that has adapted to interfere with host proteolytic systems and to modify extracellular matrix; these features likely assist the migration of Salmonella during systemic salmonellosis.

Origin of activation of Lattice Oxygen and Synergistic Interaction in Bimetal-Ionic Ce0.89Fe0.1Pd0.01O2-delta Catalyst

Flourite-type nanocrystalline Ce0.9Fe0.1O2-delta and Ce0.89Fe0.1Pd0.01O2-delta solid solutions have been synthesized by solution combustion method,'.which show higher oxygen storage/release property (OSC) compared to CeO2 and Ce0.8Zr0.2O2. Temperature programmed reduction an XPS study reveal that the presence of Pd ion in Ce0.9Fe0.1O2-delta facilitates complete reduction of Fe3+ to Fe2+ state and partial reduction of Ce4+ to Ce3+ state at.temperatures as low as 105 degrees C compared to 400 degrees C for monometal-ionic Ce0.9Fe0.1O2-delta. Fe3+ ion is reduced to Fe2+ and not to Feo due to favorable redox potential for Ce4+ + Fe2+ -> Ce3+ + Fe3+ reaction. Using first-principles density functional theory calculation we determine M-O (M = Pd, Fe, Ce) bond lengths, and find that bond lengths vary from shorter (2.16 angstrom) to longer (2.9 angstrom) bond distances compared to mean Ce-O bond distance of 2.34 angstrom. for CeO2. Using these results in bond valence analysis, we show that oxygen with bond valences as low as -1.55 are created, leading to activation of lattice oxygen in the bimetal ionic catalyst. Temperatures of CO oxidation and NO reduction by CO/H-2 are lower with the bimetalionic Ce0.89Fe0.1Pd0.01O2-delta catalyst compared to monometal-ionic Ce0.9Fe0.1O2-delta and Ce0.99Pd0.01O2-delta catalysts. From XPS studies of Pd impregnated on CeO2 and Fe2O3 oxides, we show that the synergism leading to low temperature activation of lattice oxygen in bimetal-ionic catalyst Ce0.89Fe0.1Pd0.01O2-delta is due to low-temperature reduction of Pd2+ to Pd-0, followed by Pd-0 + 2Fe(3+) -> Pd2+ + 2Fe(2+), Pd-0 + 2Ce(4+) -> Pd2+ + 2Ce(3+) redox reaction.

Sequence Specific Interaction between Promoter DNA and Escherichia coli RNA Polymerase: Comparative Thermodynamic Analysis with One Immobilized Partner

Sequence specific interaction between DNA and protein molecules has been a subject of active investigation for decades now. Here, we have chosen single promoter containing bacteriophage Delta D-III T7 DNA and Escherichia coli RNA polymerase and followed their recognition at the air-water interface by using the surface plasmon resonance (SPR) technique, where the movement of one of the reacting species is restricted by way of arraying them on an immobilized support. For the Langmuir monolayer studies, we used a RNA polymerase with a histidine tag attached to one of its subunits, thus making it an xcellent substrate for Ni(II) ions, while the SPR Studies were done using biotin-labeled DNA immobilized on a streptavidin-coated chip. Detailed analysis of the thermodynamic parameters as a function of concentration and temperature revealed that the interaction of RNA polymerase with T7 DNA is largely entropy driven (83 (+/- 12) kcal mol(-1)) with a positive enthalpy of 13.6 (+/- 3.6) kcal mol(-1), The free energy of reaction determined by SPR and Langmuir-Blodgett technique was -11 (+/- 2) and -15.6 kcal mol(-1), respectively. The ability of these methods to retain the specificity of the recognition process was also established.

An extragenic suppressor of prp24-1 defines genetic interaction between PRP24 and PRP21 gene products of Saccharomyces cerevisiae

The temperature-sensitive prp24-1 mutation defines a gene product required for the first step in pre-mRNA splicing. PRP24 is probably a component of the U6 snRNP particle. We have applied genetic reversion analysis to identify proteins that interact with PRP24. Spontaneous revertants of the temperature-sensitive (ts) prp24-1 phenotype were analyzed for those that are due to extragenic suppression. We then extended our analysis to screen for suppressors that confer a distinct conditional phenotype. We have identified a temperature-sensitive extragenic suppressor, which was shown by genetic complementation analysis to be allelic to prp21-1. This suppressor, prp21-2, accumulates pre-mRNA at the non-permissive temperature, a phenotype similar to that of prp21-1. prp21-2 completely suppresses the splicing defect and restores in vivo levels of the U6 snRNA in the prp24-1 strain. Genetic analysis of the suppressor showed that prp21-2 is not a bypass suppressor of prp24-1. The suppression of prp24-1 by prp21-2 is gene specific and also allele specific with respect to both the loci. Genetic interactions with other components of the pre-spliceosome have also been studied. Our results indicate an interaction between PRP21, a component of the U2 snRNP, and PRP24, a component of the U6 snRNP. These results substantiate other data showing U2-U6 snRNA interactions.