Differential minor groove interactions between DNA polymerase and sugar backbone of primer and template strands

DNA polymerases are the key enzymes for DNA synthesis involved in DNA replication, recombination, and repair. These enzymes undergo manifold contacts with the primer-template-substrates reaching up to several nucleotide pairs beyond the catalytic centre. To evaluate these enzyme contacts with the DNA substrates we applied novel synthetic steric probes in functional studies. We found that through application of the these probes valuable insights into DNA polymerase function can be gained, which might be useful for the design of new DNA polymerase-based nucleotide variation detection strategies.     

Solute-solvent interactions probed by intermolecular NOEs

Nuclear Overhauser effects arising from the interactions of spins of solvent molecules with spins of a solute should reveal the "exposure" of solute spins to collisions with solvent. Such intermolecular NOEs could, therefore, provide information regarding conformation or structure of the solute. Determinations of solute-solvent NOEs of 1,3-di-tert-butylbenzene in solvents composed of perfluoro-tert-butyl alcohol, tetramethylsilane, and carbon tetrachloride have been carried out. A crude, but apparently reliable, method for prediction of intermolecular solvent-solute NOEs based on hard (noninteracting) spheres was developed. Comparison of experimental to predicted NOEs indicates that tetramethylsilane interacts with the solute according to the model. By contrast, intermolecular NOE data indicate attractive interactions between the solute and perfluoro-tert-butyl alcohol. All NOE results and the corresponding predictions confirm that proton H2 of the solute is protected by the flanking tert-butyl groups from interactions with solvent molecules.     

Mucin-electrolyte interactions at the solid-liquid interface probed by QCM-D

The interaction between mucin and ions has been investigated by employing the quartz crystal microbalance technique with measurement of energy dissipation. The study was partially aimed at understanding the adsorption of mucin on surfaces with different chemistry, and for this purpose, surfaces exposing COOH, OH, and CH(3) groups were prepared. Mucin adsorbed to all three types of functionalized gold surfaces. Adsorption to the hydrophobic surface and to the charged hydrophilic surface (COOH) occured with high affinity despite the fact that in the latter case both mucin and the surface were negatively charged. On the uncharged hydrophilic surface exposing OH groups, the adsorption of mucin was very low. Another aim was to elucidate conformational changes induced by electrolytes on mucin layers adsorbed on hydrophobic surfaces from 30 mM NaNO(3). To this end, we investigated the effect of three electrolytes with increasing cation valance: NaCl, CaCl(2) and LaCl(3). At low NaCl concentrations, the preadsorbed layer expands, whereas at higher concentrations of NaCl the layer becomes more compact. This swelling/compacting of the mucin layer is fully reversible for NaCl. When the mucin layer instead is exposed to CaCl(2) or LaCl(3), compaction is observed at 1 mM. For CaCl(2), this process is only partially reversible, and for LaCl(3), the changes are irreversible within the time frame of the experiment. Finally, mucin interaction with the DTAB cationic surfactant in an aqueous solution of different electrolytes was evaluated with turbidimetry measurements. It is concluded that the electrolytes used in this work screen the association between mucin and DTAB and that the effect increases with increasing cation valency.     

Electrostatic-assembly metallized nanoparticles network by DNA template

Eighteen-nanometer gold and 3.5-nm silver colloidal particles closely packed by cetyltrimethylammonium bromide (CTAB) to form its positively charged shell. The DNA network was formed on a mica substrate firstly. Later, CTAB-capped gold or silver colloidal solutions were cast onto DNA network surface. It was found that the gold or silver nanoparticles metallized networks were formed owing to the electrostatic-driven template assembling of positive charge of CTAB-capped gold and silver particles on the negatively charged phosphate groups of DNA molecules by the characterizations of AFM, XPS and UV-vis. This method may provide a novel and simple way to studying nanoparticles assembly conjugating DNA molecules and offer some potential promising applications in nanocatalysis, nanoelectronics, and nanosensor on the basis of the fabricated metal nanoparticles network.     

Environmental effects on the enhancement in natural and damaged DNA nucleobase acidity because of discrete hydrogen-bonding interactions

The present study uses density functional theory to carefully consider the effects of the environment on the enhancement in (natural and damaged) DNA nucleobase acidities because of multiple hydrogen-bonding interactions. Although interactions with one small molecule can increase the acidity of the nucleobases by up to 60 kJ mol-1 in the gas phase, the maximum increase in enzymatic-like environments is expected to be approximately 40 kJ mol-1, which reduces to approximately 30 kJ mol-1 in water. Furthermore, the calculated (simultaneous) effects of two, three, or four molecules are increasingly less than the sum of the individual (additive) effects with an increase in the number and acidity of the small molecules bound or the dielectric constant of the solvent. Regardless of these trends, our calculations reveal that additional hydrogen-bonding interactions will have a significant effect on nucleobase acidity in a variety of environments, where the exact magnitude of the effect depends on the properties of the small molecule bound, the nucleobase binding site, and the solvent. The maximum increase in nucleobase acidity because of interactions with up to four small molecules is approximately 80 kJ mol-1 in enzymatic-like environments (or 65 kJ mol-1 in water). These results suggest that hydrogen-bonding interactions likely play an important role in many biological processes by changing the physical and chemical properties of the nucleobases.     

Divalent metal ion-peptide interactions probed by electron capture dissociation of trications

Electron capture dissociation (ECD) of the peptide Substance P (SubP) complexed with divalent metals has been investigated. ECD of [SubP + H + M]3+ (M2+ = Mg2+ -Ba2+ and Mn2+ -Zn2+) allowed observation of a larger number of product ions than previous investigations of doubly charged metal-containing peptides. ECD of Mg-Ba, Mn, Fe, and Zn-containing complexes resulted in product ions with and without the metal from cleavage of backbone amine bonds (c' and z* -type ions). By contrast, ECD of Co and Ni-containing complexes yielded major bond cleavages within the C-terminal methionine residue (likely to be the metal ion binding site). Cu-containing complexes displayed yet another behavior: amide bond cleavage (b and y'-type ions). We believe some results can be rationalized both within the hot hydrogen atom mechanism and mechanisms involving electron capture into excited states, such as the recently proposed amide superbase mechanism. However, some behavior, including formation of (cn 'M - H)+ ions for Ca-Ba, is best explained within the latter mechanisms with initial electron capture at the metal. In addition, the ECD behavior appears to correlate with the metal second ionization energy (IE2). Co and Ni (displaying sequestered fragmentation) have IE2s of 17.1 and 18.2 eV, respectively, whereas IE2s for Mg-Ba, Mn, and Fe (yielding random cleavage) are 10.0 to 16.2 eV. This behavior is difficult to explain within the hot hydrogen atom mechanism because hydrogen transfer should not be influenced by IE2s. However, the drastically different fragmentation patterns for Co, Ni, and Cu compared to the other metals can also be explained by their higher propensity for nitrogen (as opposed to oxygen) binding. Nevertheless, these results imply that directed fragmentation can be accomplished via careful selection of the cationizing agent.     

Charge transfer interactions in liquid crystalline polymers probed by fluorescence spectroscopy

Rigid-rod aromatic LC polyester with long alkyl side chains and two thermotropic LC polyesters (PET40/OBA60 and PB-10) were studied by fluorescence spectroscopy to investigate their charge transfer interactions corresponding to LC configuration and changes during phase transition.     

Supramolecular interactions probed by 13C-13C solid-state NMR spectroscopy

We present a robust solid-state NMR approach for the accurate determination of molecular interfaces in insoluble and noncrystalline protein-protein complexes. The method relies on the measurement of intermolecular (13)C-(13)C distances in mixtures of [1-(13)C]glucose- and [2-(13)C]glucose-labeled proteins. We have applied this method to Parkinson's disease-associated α-synuclein fibrils and found that they are stacked in a parallel in-register arrangement. Additionally, intermolecular distance restraints for the structure determination of the fibrils at atomic resolution were measured.     

Intermolecular interactions in electroactive thiol monolayers probed by linear scan voltammetry

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Electrostatic interactions in protein adsorption probed by comparing lysozyme and succinylated lysozyme

In this paper, we present a nanoscale study of the supramolecular structure of the dehydrogenate polymer (ZL-DHP) lignin model compound. The combination of near-field scanning optical microscopy (NSOM or SNOM) and atomic force microscopy (AFM) has been utilized to explore physicochemical properties of the lignin model compound on a scale ranging from individual macromolecules to globular supramolecular assemblies. By utilizing NSOM in transmission mode, the optical inhomogeneity in the lignin supramolecular structure has been observed for the first time. In particular, the transmission-mode NSOM images reveal a combination of hollow and layered supramolecular globular structure in the lignin model compound. Through the paired use of TappingMode and pulsed-mode AFM, we have also confirmed the existence of regions with different rheological properties on the single lignin model compound supramolecular assembly.     

Mechanistic aspects of protein/material interactions probed by atomic force microscopy

Physicochemical studies on the mechanisms of protein adsorption onto solid material surfaces have been extensively performed so far, mainly based on the analysis of factors such as the equilibrium adsorbed amount (adsorption isotherms), time-dependent change of adsorbed amount (adsorption kinetics), and conformational change of adsorbed protein. However, direct understanding of the strength of the molecular interaction between protein and the material surface has not been established yet. For this issue, the force measurement techniques of an atomic force microscope (AFM) using a protein-modified probe tip are recently becoming powerful tools to analyze the actual interaction forces between protein and material surfaces. In this mini review, we discuss the characteristics and interpretation of the AFM force-versus-distance curves (f–d curves) obtained with the protein-modified probe tip, and the relationship between the forces measured from the f–d curves and the driving forces in the natural process of protein adsorption. Relative degrees of each of the following contributions which determine the character of protein adsorption are clarified: (1) the intrinsic protein/material forces mediated by solvent, (2) the thermodynamic stability of protein/material adhesion interface, and (3) diffusion force of protein molecules. Within these driving forces, the latter two in particular are confirmed to play essential roles in determining the character of protein adsorption, based on the profiles of f–d curves.     

Conformational transition of DNA bound to Hfq probed by infrared spectroscopy

Hfq is a bacterial protein involved in RNA metabolism. Besides this, Hfq's role in DNA restructuring has also been suggested. Since this mechanism remains unclear, we examined the DNA conformation upon Hfq binding by combining vibrational spectroscopy and neutron scattering. Our analysis reveals that Hfq, which preferentially interacts with deoxyadenosine rich sequences, induces partial opening of dA-dT sequences accompanied by sugar repuckering of the dA strand and hence results in a heteronomous A/B duplex. Sugar repuckering is probably correlated with a global dehydration of the complex. By taking into account Hfq's preferential binding to A-tracts, which are commonly found in promoters, potential biological implications of Hfq binding to DNA are discussed.     

Binding mode of proflavine to DNA probed by polarized light spectroscopy

It is well known that proflavine binds to DNA. Here we investigate the binding mode of proflavine to native DNA using absorption, circular dichroism (CD), and linear dichroism (LD) spectroscopy as well as by fluorescence techniques. The observed changes of proflavine upon complexation with DNA can be summarized as a red shift and hypochromism in the absorption spectrum. The negative LD^r in the proflavine absorption region has a magnitude comparable to or larger than that of the DNA absorption region, confirming the intercalative binding mode of proflavine to DNA. Saturation of the LD spectrum in the proflavine absorption region at R = 0.25 and a decrease in the fluorescence intensity provide further evidence of intercalation. Furthermore, the coupling of electric transition of intercalated proflavine is observed. Although proflavine has been reported to position itself along the DNA stem at high [proflavine]/[DNA base] ratios, the spectral characteristics, which include a clear isosbestic point in the absorption spectrum and proportionality in the LD magnitude in the proflavine absorption region, do not show any possibility of exterior binding.     

Thermostability of salt bridges versus hydrophobic interactions in proteins probed by statistical potentials

The temperature dependence of the interactions that stabilize protein structures is a long-standing issue, the elucidation of which would enable the prediction and the rational modification of the thermostability of a target protein. It is tackled here by deriving distance-dependent amino acid pair potentials from four datasets of proteins with increasing melting temperatures (Tm). The temperature dependence of the interactions is determined from the differences in the shape of the potentials derived from the four datasets. Note that, here, we use an unusual dataset definition, which is based on the Tm values, rather than on the living temperature of the host organisms. Our results show that the stabilizing weight of hydrophobic interactions (between Ile, Leu, and Val) remains constant as the temperature increases, compared to the other interactions. In contrast, the two minima of the Arg--Glu and Arg--Asp salt bridge potentials show a significant Tm dependence. These two minima correspond to two geometries: the fork--fork geometry, where the side chains point toward each other, and the fork--stick geometry, which involves the N(epsilon) side chain atom of Arg. These two types of salt bridges were determined to be significantly more stabilizing at high temperature. Moreover, a preference for more-compact salt bridges is noticeable in heat-resistant proteins, especially for the fork--fork geometry. The Tm-dependent potentials that have been defined here should be useful for predicting thermal stability changes upon mutation.     

Characterization and mechanism of formation of tandem lesions in DNA by a nucleobase peroxyl radical

5,6-Dihydro-2'-deoxyuridin-6-yl (1) was independently generated via photolysis of 3. The radical is an analogue of the major reactive species produced from thymidine upon reaction with hydroxyl radical, which is the dominant DNA-damaging agent produced by the indirect effect of gamma-radiolysis. Under aerobic conditions, the peroxyl radical (2) derived from 1 reacts approximately 82% of the time with either the 5'- or 3'-adjacent nucleotide to produce two contiguously damaged nucleotides, known as tandem lesions. The structures and distribution of tandem lesions were investigated using probes that selectively detect abasic sites, ESI-MS/MS, and competition kinetics. In addition to 2-deoxyribonolactone, nonoxidized abasic sites were detected. 18O-Labeling verified that H2O was the source of oxygen in the abasic sites, but that O2 was the source of the oxygen in the 5,6-dihydro-6-hydroxy-2'-deoxyuridine derived from 2. ESI-MS/MS experiments, in conjunction with isotopic labeling, identified several products and provided direct evidence for peroxyl radical addition to the adjacent thymine bases. Kinetic studies revealed that peroxyl radical addition to the 5'-thymine was favored by approximately 4-5-fold over C1'-hydrogen atom abstraction from the respective deoxyribose ring, and that 2-deoxyribonolactone formation accounts for approximately 11% of the total amount of tandem lesions produced. These results suggest that tandem lesions, whose biochemical effects are largely unknown, constitute a major family of DNA damage products produced by the indirect effect of gamma-radiolysis.     

Binding of DNA purine sites to dirhodium compounds probed by mass spectrometry

The adducts formed between the antitumor active compounds [Rh(2)(O(2)CCH(3))(2)(CH(3)CN)(6)](BF(4))(2), Rh(2)(O(2)CCH(3))(4), and Rh(2)(O(2)CCF(3))(4) with DNA oligonucleotides have been assessed by matrix-assisted laser desorption ionization (MALDI) and nanoelectrospray (nanoESI) coupled to time-of-flight mass spectrometry (TOF MS). A series of MALDI studies performed on dipurine (AA, AG, GA, and GG)-containing single-stranded oligonucleotides of different lengths (tetra- to dodecamers) led to the establishment of the relative reactivity cis-[Pt(NH(3))(2)(OH(2))(2)](2+) (activated cisplatin) approximately Rh(2)(O(2)CCF(3))(4) > cis-[Pt(NH(3))(2)Cl(2)] (cisplatin) > [Rh(2)(O(2)CCH(3))(2)(CH(3)CN)(6)](BF(4))(2) > Rh(2)(O(2)CCH(3))(4) approximately Pt(C(6)H(6)O(4))(NH(3))(2) (carboplatin). The relative reactivity of the complexes is associated with the lability of the leaving groups. The general trend is that an increase in the length of the oligonucleotide leads to enhanced reactivity for Rh(2)(O(2)CCH(3))(2)(CH(3)CN)(6)](BF(4))(2) and Rh(2)(O(2)CCH(3))(4) (except for the case of [Rh(2)(O(2)CCH(3))(2)(CH(3)CN)(6)](2+), which reacts faster with the GG octamers than with the dodecamers), whereas the reactivity of Rh(2)(O(2)CCF(3))(4) is independent of the oligonucleotide length. When monitored by ESI, the dodecamers containing GG react faster than the respectiveAA oligonucleotides in reactions with Rh(2)(O(2)CCF(3))(4) and Rh(2)(O(2)CCH(3))(2)(CH(3)CN)(6)](BF(4))(2), whereas AA oligonucleotides react faster with Rh(2)(O(2)CCH(3))(4). The mixed (AG, GA) purine sequences exhibit comparable rates of reactivity with the homopurine (AA, GG) dodecamers in reactions with Rh(2)(O(2)CCH(3))(4). The observation of initial dirhodium-DNA adducts with weak axial (ax) interactions, followed by rearrangement to more stable equatorial (eq) adducts, was achieved by electrospray ionization; the Rh-Rh bond as well as coordinated acetate or acetonitrile ligands remain intact in these dirhodium-DNA adducts. MALDI in-source decay (ISD), collision-induced dissociation (CID) MS-MS, and enzymatic digestion studies followed by MALDI and ESI MS reveal that, in the dirhodium compounds studied, the purine sites of the DNA oligonucleotides interact with the dirhodium core. Ultimately, both MALDI and ESI MS proved to be complementary, valuable tools for probing the identity and stability of dinuclear metal-DNA adducts.     

Carbohydrate-protein recognition probed by density functional theory and ab initio calculations including dispersive interactions

Carbohydrate-protein recognition has been studied by electronic structure calculations of complexes of fucose and glucose with toluene, p-hydroxytoluene and 3-methylindole, the latter aromatic molecules being analogues of phenylalanine, tyrosine and tryptophan, respectively. We use mainly a density functional theory model with empirical corrections for the dispersion interactions (DFT-D), this method being validated by comparison with a limited number of high level ab initio calculations. We have calculated both binding energies of the complexes as well as their harmonic vibrational frequencies and proton NMR chemical shifts. We find a range of minimum energy structures in which the aromatic group can bind to either of the two faces of the carbohydrate, the binding being dominated by a combination of OH-pi and CH-pi dispersive interactions. For the fucose-toluene and alpha-methyl glucose-toluene complexes, the most stable structures involve OH-pi interactions, which are reflected in a red shift of the corresponding O-H stretching frequency, in good quantitative agreement with experimental data. For those structures where CH-pi interactions are found we predict a corresponding blue shift in the C-H frequency, which parallels the predicted proton NMR shift. We find that the interactions involving 3-methylindole are somewhat greater than those for toluene and p-hydroxytoluene.     

Probing essential nucleobase functional groups in aptamers and deoxyribozymes by nucleotide analogue interference mapping of DNA

Nucleotide analogue interference mapping of DNA (dNAIM) is here introduced as a new nonenzymatic interference-based approach that enables high-throughput identification of essential nucleobase functional groups in DNA aptamers and in the catalytic core of deoxyribozymes. Nucleobase-modified ribonucleotides are statistically incorporated into DNA by solid-phase synthesis, employing the 2'-OH group as a chemical tag for analysis of interference effects. This method is exemplified on an AMP-binding DNA aptamer and was further used to identify indispensable nucleobase functional groups for DNA-catalyzed RNA-ligation by the Mg(2+)-dependent deoxyribozymes 7S11 and 9DB1. dNAIM should prove broadly useful for facile structural probing of functional DNA for which active and inactive variants can be separated based on catalytic or ligand-binding activities.